Adeno-associated virus vectors

ABSTRACT

The invention provides an isolated and purified DNA molecule comprising at least one DNA segment, a biologically active subunit or variant thereof, of a circular intermediate of adeno-associated virus, which DNA segment confers increased episomal stability, persistence or abundance of the isolated DNA molecule in a host cell. The invention also provides a composition comprising at least two adeno-associated virus vectors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation under 37 C.F.R. 1.53(b) of U.S.application Ser. No. 11/058,751 filed Feb. 15, 2005, which is acontinuation under 37 C.F.R. 1.53(b) of U.S. application Ser. No.10/054,665 filed Jan. 22, 2002 (now U.S. Pat. No. 6,897,045), which is acontinuation under 37 C.F.R. 1.53(b) of U.S. application Ser. No.09/276,625, filed Mar. 25, 1999 (now U.S. Pat. No. 6,436,392), which isa continuation-in-part application claiming the benefit of U.S.provisional application Ser. No. 60/086,166 filed May 20, 1998, thedisclosures of which applications are incorporated by reference herein.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with a grant from the Government of the UnitedStates of America (Grant No. DK/HL58340 from the National Institutes ofHealth). The Government may have certain rights in the invention.

BACKGROUND OF THE INVENTION

Adeno-associated virus (AAV) is a non-pathogenic parvovirus with asingle-stranded DNA genome of 4680 nucleotides. The genome may be ofeither plus or minus polarity, and codes for two groups of genes, Repand Cap (Berns et al., 1990). Inverted terminal repeats (ITRs),characterized by palindromic sequences producing a high degree ofsecondary structure, are present at both ends of the viral genome. Whileother members of the parvovirus group replicate autonomously, AAVrequires co-infection with a helper virus (i.e., adenovirus or herpesvirus) for lytic phase productive replication. In the absence of ahelper virus, wild-type AAV (wtAAV) establishes a latent, non-productiveinfection with long-term persistence by integrating into a specificlocus on chromosome 19, AAVS1, of the host genome through aRep-facilitated mechanism (Samulski, 1993; Linden et al., 1996; Kotin etal., 1992).

In contrast to wtAAV, the mechanism(s) of latent phase persistence ofrecombinant AAV (rAAV) is less clear. rAAV integration into the hostgenome is not site-specific due to deletion of the AAV Rep gene(Ponnazhagan et al., 1997). Analysis of integrated proviral structuresof both wild type and recombinant AAV have demonstrated head-to-tailgenomes as the predominant structural forms.

rAAV has recently been recognized as an extremely attractive vehicle forgene delivery (Muzyczka, 1992). rAAV vectors have been developed bysubstituting all viral open reading frames with a therapeutic minigene,while retaining the cis elements contained in two inverted terminalrepeats (ITRs) (Samulski et al., 1987; Samulski et al., 1989). Followingtransduction, rAAV genomes can persist as episomes (Flotte et al., 1994;Afione et al., 1996; Duan et al., 1998), or alternatively can integraterandomly into the cellular genome (Berns et al., 1996; McLaughlin etal., 1988; Duan et al., 1997; Fisher-Adams et al., 1996; Kearns et al.,1996; Ponnazhagan et al., 1997). However, little is known about themechanisms enabling rAAV vectors to persist in vivo or the identity ofcellular factors which may modulate the efficiency of transduction andpersistence. Although transduction of rAAV has been demonstrated invitro in cell culture (Muzyczka, 1992) and in vivo in various organs(Kaplitt et al., 1994; Walsh et al., 1994; Conrad et al., 1996; Herzoget al., 1997; Snyder et al., 1997), the mechanisms of rAAV-mediatedtransduction remain unclear.

Moreover, while rAAV has been shown to be capable of stable, long-termtransgene expression both in vitro and in vivo in a variety of tissues,the transduction efficiency of rAAV is markedly variable in differentcell types. For example, rAAV has been reported to transduce lungepithelial cells at low levels (Halbert et al., 1997; Duan et al.,1998a), while high level, persistent transgene expression has beendemonstrated in muscle, neurons and in other non-dividing cells (Kessleret al., 1996; Fisher et al., 1997; Herzog et al., 1997; Xiao et al.,1996; Kaplitt et al., 1994; Wu et al., 1998; Ali et al., 1996; Bennettet al., 1997 Westfall et al., 1997). These tissue-specific differencesin rAAV mediated gene transfer may, in part, be due to variable levelsof cellular factors affecting AAV infectivity (i.e., receptors andco-receptors such as heparin sulfate proteoglycan, FGFR-1, and αVβ5integrin) (Summerford et al., 1998; Qing et al., 1999; Summerford etal., 1999) as well as the latent life cycle (i.e., nuclear traffickingof virus and/or the conversion of single stranded genomes to expressibleforms) (Qing et al., 1997; Qing et al., 1998).

Muscle-mediated gene transfer represents a very promising approach forthe treatment of hereditary myopathies and several other metabolicdisorders. Previous studies have demonstrated remarkably efficient andpersistent transgene expression to skeletal muscle in vivo with rAAVvectors. Applications in this model system include the treatment ofseveral inherited disorders such as Factor IX deficiency in hemophilia Band epo deficiencies (Kessler et al., 1996; Herzog et al., 1997).Although the conversion of low-molecular-weight rAAV genomes tohigh-molecular-weight concatamers has been inferred as evidence forintegration of proviral DNA in the host genome, no direct evidenceexists in this regard (Xiao et al., 1996; Clark et al., 1997; Fisher etal. 1997). Also, the molecular processes and/or structures associatedwith episomal long-term persistence of rAAV genomes, e.g., innondividing mature myofibers, remains unclear.

Thus, there is a need for rAAV vectors that have increased stabilityand/or persistence in host cells. Moreover, there is a need for vectorsuseful to express large open reading frames.

SUMMARY OF THE INVENTION

The present invention provides a recombinant adeno-associated virus(rAAV) vector comprising a nucleic acid segment formed by thejuxtaposition of sequences in the AAV inverted terminal repeats (ITRs)which are present in a circular intermediate of AAV. The circularintermediate was isolated from rAAV-infected cells by employing arecombinant AAV “shuttle” vector. The shuttle vector comprises: a) abacterial origin of replication; b) a marker gene or a selectable gene;c) a 5′ ITR; and d) a 3′ ITR. Preferably, the recombinant AAV shuttlevector contains a reporter gene, e.g., a GFP, alkaline phosphatase orβ-galactosidase gene, a selectable marker gene, e.g., anampicillin-resistance gene, a bacterial origin of replication, a 5′ ITRand a 3′ ITR. The vector is contacted with eukaryotic cells so as toyield transformed eukaryotic cells. Low molecular weight DNA (“HirtDNA”) from the transformed eukaryotic cells is isolated. Bacterial cellsare contacted with the Hirt DNA so as to yield transformed bacterialcells. Then bacterial cells are identified which express the marker orselectable gene present in the shuttle vector and which comprise atleast a portion of a circular intermediate of adeno-associated virus.Also, as described below, it was found that circularized intermediatesof rAAV impart episomal persistence to linked sequences in Hela cells,fibroblasts and muscle cells. In HeLa cells, the incorporation ofcertain AAV sequences, e.g., ITRs, from circular intermediates into aheterologous plasmid conferred a 10-fold increase in the stability ofplasmid-based vectors in HeLa cells. Unique features of thesetransduction intermediates included the in vivo circularization of ahead-to-tail monomer as well as multimer (concatamers) episomal viralgenomes with associated specific base pair alterations in the 5′ viralD-sequence. The majority of circular intermediates had a consistenthead-to-tail configuration consisting of monomer genomes (<3 kb) whichslowly converted to large multimers of >12 kb by 80 days post-infectionin muscle. Importantly, long-term transgene expression was associatedwith prolonged (80 day) episomal persistence of these circularintermediates. Thus, in vivo persistence of rAAV can occur throughepisomal circularized genomes which may represent prointegrationintermediates with increased episomal stability. Moreover, as describedbelow, co-infection with adenovirus, at high multiplicities of infection(MOI) capable of producing early adenoviral gene products, led toincreases in the abundance and stability of AAV circular intermediateswhich correlated with an elevation in transgene expression from rAAVvectors. Thus, these results demonstrate the existence of a molecularstructure involved in AAV transduction which may play a role in episomalpersistence and/or integration.

Further, these results may aid in the development of non-viral orviral-based gene delivery systems having increased efficiency. Forexample, therapeutic or prophylactic therapies in which the presentvectors are useful include blood disorders (e.g., sickle cell anemia,thalassemias, hemophilias, and Fanconi anemias), neurological disorders,such as Alzheimer's disease and Parkinson's disease, and muscledisorders involving skeletal, cardiac or smooth muscle. In particular,therapeutic genes useful in the vectors of the invention include theβ-globin gene, the γ-globin gene, the cystic fibrosis transmembraneconductance receptor gene (CFTR), the Fanconi anemia complementationgroup, a gene encoding a ribozyme, an antisense gene, a low densitylipoprotein (LDL) gene, a tyrosine hydroxylase gene (Parkinson'sdisease), a glucocerebrosidase gene (Gaucher's disease), anarylsulfatase A gene (metachromatic leukodystrophies) or genes encodingother polypeptides or proteins. Also within the scope of the inventionis the inclusion of more than one gene in a vector of the invention,i.e., a plurality of genes may be present in an individual vector.Further, as a circular intermediate may be a concatamer, each monomer ofthat concatamer may comprise a different gene.

For viral-based delivery systems, helper-free virus can be prepared (seeWO 95/13365) from circular intermediates or vectors of the invention.Alternatively, liposomes, plasmid or virosomes may be employed todeliver a vector of the invention to a host or host cell.

The increased persistence of circular intermediates or vectors havingone or a plurality of ITRs may be due to the primary and/or secondarystructure of the ITRs. The primary structure of a consensus sequence(SEQ ID NO:3) of ITRs formed by the juxtaposition and physical(phosphodiester bond) linkage of ITRs from AAV is shown in FIG. 2C.However, as described hereinbelow, each ITR sequence may be incomplete,i.e., the ITR may be a subunit or portion of the full length ITRspresent in the consensus sequence. Moreover, preferably, an isolated DNAsegment of the invention is not the 165 bp double DD sequence (SEQ IDNO:7) disclosed in U.S. Pat. No. 5,478,745, referred to as a “doublesequence”.

Moreover, the formation, persistence and/or abundance of moleculeshaving the ITR sequences of the invention may be modulated by helpervirus, e.g., adenoviral proteins and/or host cell proteins. Thus, thecircular intermediates or vectors of the invention may be useful toidentify and/or isolate proteins that bind to the ITR sequences presentin those molecules.

Therefore, the present invention provides an isolated and purified DNAmolecule comprising at least one DNA segment, a biologically activesubunit or variant thereof, of a circular intermediate ofadeno-associated virus, which DNA segment confers increased episomalstability, persistence or abundance of the isolated DNA molecule in ahost cell. Preferably, the DNA molecule comprises at least a portion ofa left (5′) inverted terminal repeat (ITR) of adeno-associated virus.Also preferably, the DNA molecule comprises at least a portion of aright (3′)-inverted terminal repeat of adeno-associated virus. Theinvention also provides a gene transfer vector, comprising: at least onefirst DNA segment, a biologically active subunit or variant thereof, ofa circular intermediate of adeno-associated virus, which DNA segmentconfers increased episomal stability or persistence of the vector in ahost cell; and a second DNA segment comprising a gene. Preferably, thesecond DNA segment encodes a therapeutically effective polypeptide. Thefirst DNA segment comprises ITR sequences, preferably at least about100, more preferably at least about 300, and even more preferably atleast about 400, bp of adeno-associated virus sequence. A preferredvector of the invention is a plasmid.

Thus, the vector of the invention is useful in a method of deliveringand/or expressing a gene in a host cell, to prepare host cells havingthe vector(s), and in the preparation of compositions comprising suchvectors. To deliver the gene to the host cell, a recombinant adenovirushelper virus may be employed.

As described hereinbelow, the tibialis muscle of mice was co-infectedwith rAAV Alkaline phosphatase (Alkphos) and GFP encoding vectors. TheGFP shuttle vector also encoded ampicillin resistance and a bacterialorigin of replication to allow for bacterial rescue of circularintermediates in Hirt DNA from infected muscle samples. There was a timedependent increase in the abundance of rescued plasmids encoding bothGFP and Alkphos that reached 33% of the total circular intermediates by120 days post-infection. Furthermore, these large circular concatamerswere capable of expressing both GFP and Alkphos encoded transgenesfollowing transient transfection in cell lines. Thus, concatamerizationof AAV genomes in vivo occurs through intermolecular recombination ofindependent monomer circular viral genomes. Therefore, a plurality ofDNA segments, each in an individual rAAV vector, may be delivered so asto result in a single DNA molecule having a plurality of the DNAsegments. For example, one rAAV vector comprises a first DNA segmentcomprising a 5′ ITR linked to a second DNA segment comprising a promoteroperably linked to a third DNA segment comprising a first open readingframe linked to a fourth DNA segment comprising a 3′ ITR. A second rAAVvector comprises a first DNA segment comprising a 5′ ITR linked to asecond DNA segment comprising a promoter operably linked to a third DNAsegment comprising a second open reading frame linked to a fourth DNAsegment comprising a 3′ ITR.

In another embodiment, one rAAV vector comprises a first DNA segmentcomprising a 5′ ITR linked to a second DNA segment comprising a promoteroperably linked to a third DNA segment comprising the 5′ end of an openreading frame linked to fourth DNA segment comprising a 5′ splice sitelinked to a fifth DNA segment comprising a 3′ ITR. The second rAAVvector comprises a first DNA segment comprising a 5′ ITR linked to asecond DNA segment comprising a 3′ splice site linked to a third DNAsegment comprising the 3′ end of the open reading frame linked to afourth DNA segment comprising a 3′ ITR. Preferably, the second and thirdDNA segments together comprise DNA encoding, for example, CTFR, factorVIII, dystrophin, or erythropoietin. Also preferably, the second DNAsegment comprises the endogenous promoter of the respective gene, e.g.,the epo promoter.

Thus, the invention provides a composition comprising: a firstadeno-associated virus vector comprising linked DNA segments and atleast a second adeno-associated virus comprising linked DNA segments.The linked DNA segments of the first vector comprise: a first DNAsegment comprising a 5′ ITR; a second DNA segment comprising at least aportion of an open reading frame operably linked to a promoter, whereinthe DNA segment does not comprise the entire open reading frame; a thirdDNA segment comprising a splice donor site; and iv) a fourth DNA segmentcomprising a 3′ ITR. The linked DNA segments of the second vectorcomprise a first DNA segment comprising a 5′ ITR; a second DNA segmentcomprising a splice acceptor site; a third DNA segment comprising atleast a portion of an open reading frame which together with the secondDNA segment of the first vector encodes a full-length polypeptide; and afourth DNA segment comprising a 3′ ITR. Preferably, the second DNAsegment of the first vector comprises a first exon of a gene comprisingmore than one exon and the third DNA segment of the second vectorcomprises at least one exon of a gene that is not the first exon.

The invention also provides a method to transfer and express apolypeptide in a host cell. The method comprises contacting the hostcell with at least two rAAV vectors. One rAAV vector comprises a firstDNA segment comprising a 5′ITR linked to a second DNA segment comprisinga promoter operably linked to a third DNA segment comprising a firstopen reading frame linked to a fourth DNA segment comprising a 3′ ITR. Asecond rAAV vector comprises a first DNA segment comprising a 5′ ITRlinked to a second DNA segment comprising a promoter operably linked toa third DNA segment comprising a second open reading frame linked to afourth DNA segment comprising a 3′ITR. Alternatively, one rAAV vectorcomprises a first DNA segment comprising a 5′ITR linked to a second DNAsegment comprising a promoter operably linked to a third DNA segmentcomprising the 5′ end of an open reading frame linked to fourth DNAsegment comprising a 5′ splice site linked to a fifth DNA segmentcomprising a 3′ ITR. The second rAAV vector comprises a first DNAsegment comprising a 5′ ITR linked to a second DNA segment comprising a3′ splice site linked to a third DNA segment comprising the 3′ end ofthe open reading frame linked to a fourth DNA segment comprising a3′ITR. The host cell is preferably contacted with both of the vectors,concurrently, although it is envisioned that the host cell may becontacted with each vector at a different time relative to the contactwith the other vector(s).

Also provided is a method in which the composition of the invention isadministered to the cells or tissues of an animal. For example, rAAVvectors have shown promise in transferring the CFTR gene into airwayepithelial cells of animal models and nasal sinus of CF patients.However, high level expression of CFTR has not been achieved due to thefact that AAV cannot accommodate the full-length CFTR gene together witha potent promoter. A number of studies have tried to optimizerAAV-mediated CFTR expression by utilizing truncated or partiallydeleted CFTR genes together with stronger promoters. However, it iscurrently unknown what effect deletions within the CFTR gene may have oncomplementation of bacterial colonization defects in the CF airway.Therefore, the present invention includes the administration to ananimal of a composition of the invention comprising at least two rAAVvectors which together encode CTFR. The present invention is useful toovercome the current size limitation for transgenes within rAAV vectors,and allows for the incorporation of a larger transcriptional regulatoryregion, e.g., a stronger heterologous promoter or the endogenous CFTRpromoter.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Structure of proviral shuttle vector and the predicted structureof rAAV circular intermediate monomers. With the aid of a rAAVcis-acting plasmid, pCisAV.GFP3ori (Panel A), AV.GFP3ori recombinantvirus was produced (Panel B). This vector encoded a GFP transgenecassette, an ampicillin resistance gene (amp), and a bacterialreplication origin (ori). The predominant form of circular intermediatesisolated following transduction of Hela cells with AV.GFP3ori consistedof head-to-tail monomers (Panels C and D).

FIG. 2. Structural analysis of rAAV circular intermediates in Hela cells(agarose gel, left; Southern blot, right). Circular rAAV intermediateclones isolated from AV.GFP3ori infected Hela cells were analyzed bydiagnostic restriction digestion with AseI, SphI, and PstI together withSouthern blotting against ITR, GFP, and Stuffer ³²P-labeled probes. Inpanel A, four clones representing the diversity of intermediates found(p190, p333, p280, and p345) gave a diagnostic PstI (P) restrictionpattern (3 kb and 1.7 kb bands) consistent with a circular monomer ormultimer intact genome. SphI (S) digestion demonstrated existence of asingle ITR (p190), two ITRs in a head-to-tail orientation (p333 andp280), and three ITRs (p345) in isolated circular intermediates. Therestriction pattern of pCisAV.GFP3ori (U; uncut, P; PstI cut, S; SphIcut) and 1 kb DNA ladder (L) are also given for comparison. Oneadditional circular form (p340) was repetitively seen and had anunidentifiable structure which lacked intact ITR sequences. Circularconcatamers were identified by partial digestion with AseI for clonesp280 (dimer) and p333 (monomer) as is shown in Panel B. Sequenceanalysis (Panel C) of six clones with identical restriction patterns top333 (Panel A) was performed using primers (indicated by arrows)juxtaposed to the partial p5 promoter (dotted line) and ITRs (solidline) (SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, and SEQ ID NO: 13). The top sequence represents the proposedhead-to-tail structure of intact ITR arrays with alignment of sequencederived from individual clones. The junction of the inverted ITRs ismarked by inverted arrowheads (at 251 bp). Several consistent bp changes(shaded) were noted in the 5′ITR D-sequence (boxed) within four clones(p79, p81, p87, and p88). All bp changes are indicated in lower caseletters.

FIG. 3. Adenovirus augments AAV circular intermediate formation in Helacells. Infection of Hela cells with increasing doses (0, 500, and 5000particles/cell) of recombinant E1-deleted adenovirus (Ad.CMVlacZ) leadsto substantial expression of E2a 72 kd DNA Binding Protein, asdemonstrated by indirect immunofluorescent staining for DBP at 72 hourspost-infection (Panel A). Co-infection of Hela cells with Ad.CMVlacZ(5000 particles/cell) and AV.GFP3ori (1000 DNA particles/cell) led tosubstantial augmentation of rAAV GFP transgene expression (Panel B).Augmentation in rAAV GFP transgene expression in the presence ofincreasing amounts (0, 500, 5000 and 10000 particles/cell) ofrecombinant Ad.CMVlacZ was quantified by FACS analysis at 72 hourpost-infection (Panel C). Results demonstrate the mean (+/−SEM) for twoexperiments performed in duplicate. In addition, an aliquot of cells wassplit (1:10) at the time of FACS analysis and GFP colony forming units(CFU) per 10× field were quantified at 6 days (CPE denotes significantcytopathic effects at an adenoviral MOI of 10,000 particles/cell and wasnot quantified for GFP colonies). Hirt DNAs from AV.GFP3ori (1000 DNAparticles/cell) infected Hela cells with or without co-infection withAd.CMVlacZ (5,000 particles/cell) were used to transform E. coli. Thetotal number of ampicillin-resistant bacterial CFU (Panel D) and totalnumber of head-to-tail circular intermediates CFU (Panel E) are givenfor a representative experiment. Greater than 20 clones for each timepoint were evaluated by Southern blot (see FIG. 2 for detail). Zero hourcontrols were performed by mixing an equivalent amount of AV.GFP3orivirus as used in experiments with mock infected cellular lysates priorto Hirt purification. Panel F depicts the abundance of head-to-tailcircular intermediates as a percentage of total ampicillin-resistantbacterial CFU isolated from Hirt DNA.

FIG. 4. Formation of rAAV head-to-tail circular intermediates followingin vivo transduction of muscle. The tibialis anterior muscle of 4-5 weekold C57BL/6 mice were infected with AV.GFP3ori (3×1010 particles) inHEPES buffered saline (30 μl). GFP expression (Panel A) was analyzed bydirect immunofluorescence of freshly excised tissues and/or informalin-fixed cryopreserved tissue sections in four independentlyinjected muscles harvested at 0, 5, 10, 16, 22 and 80 dayspost-infection. GFP expression was detected at low levels beginning at10 days and was maximum at 22 days post-infection. Expression remainedstable to 80 days at which time greater than 50% of the tissue waspositive (see 80 day tissue cross section counter stained with propidiumiodide, panel A). Hirt DNA was isolated from muscle samples at each ofthe various time points and after points was used to transform E. coli.Rescued plasmids (p439, p16, p17) were analyzed by Southern blotting inPanel B showing an agarose gel on left and ITR probed blot on right.U:uncut, P:PstI cut, and S:SphI cut. The schematic drawing of the mostpredominant type of head-to-tail circular AAV intermediate plasmidsrescued from bacteria is given in the right of Panel B and shows thestructure of p17 as an example. Other typical clones included those withless than two ITRs as shown for p16. SphI digestion of p16 and p17plasmids released ITR hybridizing fragments of approximately 140 and 300bp, respectively. The slightly lower mobility then predicted for theseITR fragments likely represents anomalous migration due to the highsecondary structure of inverted repeats within ITRs. Sequence analysisof p17 and p16 using nested primers to 5′ and 3′-ITRs also confirmed theITR orientations shown to the right of the gel. Additional restrictionenzyme analyses to determine this structure included double and singledigests with SphI, PstI, AseI, and/or SmaI. An example of an atypicalclone (p439) rescued from bacteria with unknown structure is also shown.

FIG. 5. Frequency of circular intermediate formation in muscle followingtransduction with rAAV. Hirt DNAs isolated from rAAV infected tibialismuscle were used to transform E. coli and the rescued plasmids analyzedby Southern blotting (greater than 20 clones were analyzed from at leasttwo independent muscle samples for each time point). The averages oftotal head-to-tail circular intermediate clones (line) and ampicillinresistant bacterial clones (bar) isolated from each tibialis anteriormuscle at 0, 5, 10, 16, 22 and 80 days post-infection are summarized inPanel A. Only plasmids which contained 1-2 ITRs were included in theestimation of total head-to-tail circular intermediates. Plasmids whichdemonstrated an absence of ITR hybridizing SphI fragments (between 150to 300 bp) were omitted from the calculations. Panel B demonstrates thediversity of ITR arrays found in head-to-tail circular intermediates at80 days post-infection. This panel depicts a Southern blot probed withITR sequences and represents circular intermediates with 1-3 ITRs. SphIfragments which hybridize to ITR probes indicate the size of invertedITR arrays (marked by arrows to right of gel). Additional restrictionenzyme analysis was used to determine the structure of monomer andmultimer circular intermediates. Examples are shown for two multimer(p136 and p143) circular intermediates which contain approximately threeAAV genomes. Undigested plasmids of p136 and p143 migrate greater than12 kb and is contrasted to the most predominant form of head-to-tailundigested circular intermediates at 22 days which migrate at 2.5 kb.The digestion pattern of p136 is consistent with a uniform head-to-tailconfiguration of three genomes which is indistinguishable from digestionpatterns of p139 which contains one circularized genome (undigested p139migrates at 2.5 kb, data not shown, also see examples p17 in FIG. 4). Incontrast, p136 depicts a more complex head-to-tail multimer circularintermediate which has various deletions and duplications within the ITRarrays. Predicted structure of five representative intermediates isschematically shown in Panel C.

FIG. 6. Molecular size of circular intermediates in muscle. Hirt DNAfrom AV.GFP3ori infected muscle was size fractionated by electrophoresisand various molecular weight fractions transformed into E. coli. Resultsdemonstrate the abundance of circular intermediates at each of the givenmolecular weights at 22 and 80 days post-infection with the rAAV shuttlevector. Structure of circular intermediates were confirmed by Southernblot restriction analysis.

FIG. 7. Head-to-tail circular intermediates demonstrate increasedstability of GFP expression following transient transfection in Helacells. Subconfluent monolayers of Hela cells were co-transfected withp81, p87, or pCMVGFP and pRSVlacZ as an internal control fortransfection efficiency as described in the methods. Panel Ademonstrates the expansion of GFP clones after one passage (arrows).Quantification of clone size and numbers are shown in Panel B. Clonesize represents the mean raw values while clone numbers are normalizedfor transfection efficiency as determined by X-gal staining forpRSVlacZ. The data at the top of bar graph values for each construct inPanel B represents quantification of GFP clones after second passage(also normalized for transfection efficiency). Results indicate the mean(+/−SEM) of duplicate experiments with greater than 20 fields quantifiedfor each experimental point. The persistence of transfected p81 andpCMVGFP plasmid DNA at passage-7 post-transfection was evaluated bygenomic Southern blot of total cellular DNA hybridized against³²P-labeled GFP probe (Panel C, results from two independenttransfections are shown). U:uncut, C:PstI cut. The migration of uncutdimer and monomer plasmids forms are marked on the left. PstI digestionof the plasmids results in bands at 4.7 kb (pCMVGFP, single PstI site inplasmid) and 1.7 kb (p81, two PstI sites flanking the GFP gene). Todetermine whether the head-to-tail ITR array within circularintermediates was responsible for increases in the persistence of GFPexpression, the head-to-tail ITR DNA element was subcloned into the pGL3luciferase plasmid to generate pGL3(ITR). Results in Panel D compare theextent of luciferase transgene expression following transfection withpGL3 and pGL3(ITR) at 10 days (passage-2) post-transfection. Results arethe mean (+/−SEM) for triplicate experiments and are normalized fortransfection efficiency using a dual renilla luciferase reporter vector(pRLSV40, Promega).

FIG. 8. Identification of adenoviral genes responsible for augmentationof AAV circular intermediate formation. Hela cells were infected withAV.GFP3ori (1000 DNA particles/cell) in the presence of wtAd5, dl802(E2a-deleted), and dl 1004 (E4-deleted) adenovirus (at the indicatedMOIs). Total number of head-to-tail circular intermediates from Hirt DNAand the level of augmentation of GFP transgene expression (as determinedby FACS) was quantified at 24 hours post-infection (Panel A). Resultsare the average of duplicate experiments. Panel B depicts results fromSouthern blot analysis of Hirt DNA following hybridization to a GFPP³²-labeled probe. DNA loads were 10% of the total Hirt yield from a 35mm plate of Hela cells. Infections were carried out identically to thatdescribed for Panel A. Arrows mark replication form concatamers(Rf_(c)), dimers (Rf_(d)), monomers (Rf_(m)), and single-stranded AAVgenomes (ssDNA).

FIG. 9. Model for independent mechanistic interactions of adenoviruswith lytic and latent phase aspects of the AAV life cycle. Theadenoviral E4 gene has been shown to augment the level of rAAV secondstrand synthesis giving rise to replication form dimers (Rf_(d)) andmonomers (Rf_(m)) (FIG. 8B). This augmentation leads to substantialincreases in transgene expression from rAAV vectors and most closelymirrors lytic phase replication of wtAAV as head-to-head andtail-to-tail concatamers. In contrast, E4 expression inhibits theformation of head-to-tail circular intermediates of AAV. Hence, itappears that increases in the amount of Rf_(d) and Rf_(m) doublestranded DNA genomes does not increase the extent of circularintermediate formation. Such findings suggest that conversion of Rf_(m)and Rf_(d) to circular intermediates does not likely occur andimplicates two mechanistically distinct pathway for their formation. Insupport of this hypothesis, adenoviral E2a gene expression does notenhance the formation of Rf_(m) and Rf_(d) genomes but rather increasethe abundance and/or stability of head-to-tail circular intermediates.Furthermore, in the absence of E4, E2a gene expression does not lead toaugmentation of rAAV transgene expression. Since circular intermediateshave increased episomal stability in muscle and in Hela cells, thismolecular structure may be important in the latent phase of AAVpersistence. Alternatively, these circular intermediates may representpre-integration complexes as previously hypothesized for Rep facilitatedintegration. In the absence of Rep, circular intermediates mayaccumulate episomally in rAAV infected cells. In summary, these findingssupport the notion that adenovirus may modulate both latent and lyticaspects of the AAV life cycle.

FIG. 10. Individual chemical sequence of SphI fragments from p81 (A; SEQID NO:4), p79 (B; SEQ ID NO:5), and p1202 (C; SEQ ID NO:6) AAV circularintermediates. The ends of the sequence (underlined) represent SphIrestriction enzyme sites within head-to-tail circular AAV genomes clonedwith the AV-GFP3ori shuttle virus.

FIG. 11. Chemical sequence homology of three AAV circular intermediates(SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6) with various conformations ofITR arrays. Diversity in ITR arrays are evident from the non-conservedbases marked in lower case. The ends of the sequence (underlined)represent SphI restriction enzyme sites within head-to-tail circular AAVgenomes cloned with the AV.GFP3ori shuttle virus.

FIG. 12A. Palindromic repeat structure derived from chemical sequencingof AAV circular intermediate isolate p81. Secondary structure of thesense strand is depicted in the top box with plasmid reference givenbelow.

FIG. 12B. Palindromic repeat structure derived from chemical sequencingof AAV circular intermediate isolate p79. Secondary structure of thesense strand is depicted in the top box with plasmid reference givenbelow.

FIG. 12C. Palindromic repeat structure derived from chemical sequencingof AAV circular intermediate isolate p79. Secondary structure of thesense strand is depicted in the top box with plasmid reference givenbelow.

FIG. 13. Persistence of GFP expression in developing Xenopus embryosmicroinjected with AAV circular intermediate isolate p81. The extent ofGFP fluorescence in tadpoles reflects the stability of episomal orintegrated microinjected plasmids. Bright field image on the left is ofthe p81 injected embryo. The p81 injected embryo depicts fluorescence innearly all cells by one week post-injection. In contrast, a mosaicpattern of expression in a minority of cells in pCisAV.GFPori injectedembryos. The pCisAV.GFPori plasmid contains the identical promotersequences driving GFP gene expression and two ITRs separated by stuffersequence. These findings demonstrate that specific structuralcharacteristics found within AAV circular intermediates are responsiblefor increased persistence of transgene expression.

FIG. 14. Mechanistic scheme for determining pathways for rAAV circularconcatamer formation. The two independent vectors used in these studies,AV.Alkphos and AV.GFP3.ori, are shown in Panel A. Restriction sitesimportant in the structural analysis of circular intermediates are alsoshown. In Panel B, a schematic representation of two potential modelsfor circular concatamer formation is depicted, along with the methods toexperimentally differentiate which of these processes is active inmuscle. Following co-infection of the tibialis muscle with AV.Alkphosand AV.GFP3.ori, all subsequently rescued plasmids arise solely fromcircular intermediates containing AV.GFP3ori genomes. If rollingcircular replication is the sole mechanism of concatamerization, onlyGFP expressing plasmids should be rescued. In contrast, ifintermolecular recombination between independently formed monomercircular intermediates is the mechanism of concatamerization, both GFPand GFP/Alkphos expressing plasmids should be rescued.

FIG. 15. Co-infection of tibialis muscle of mice with AV.Alkphos andAV.GFP3ori. Transgene expression of rAAV infected tibialis muscle wasdetermined at 14, 35, 80 (Panels A and A′), and 120 (Panels B-D) daysfollowing co-infection with 5×10⁹ DNA particles each of AV.Alkphos andAV.GFP3ori. The time course of transgene expression started around 14days and peaked by 35-80 days. The extent of co-infection of myofiberswith both Alkphos and GFP rAAV was determined in serial sections of 80and 120 day post-infection muscle samples. Panels A-C represent GFPfluorescence of formalin fixed, cryoprotected sections, while panelsA′-C′ depict the histochemical staining for Alkaline phosphatase inadjacent serial sections. A short staining time (7 minutes) wasnecessary to observe variation in staining levels for comparison to GFP.It was found that longer staining times (30 minutes) saturated theAlkphos signal. The boxed region in panels B and B′ are enlarged inpanels C and C′, respectively. A more precise correlation of GFP andAlkphos staining in myofibers is given in Panel D in whichco-localization of GFP and Alkphos expression was examined in the samesection of a 120 day post-infected sample. This was performed byphotographing the GFP fluorescent image prior to staining for Alkphosactivity. The left panel of D shows a high power Nomarskiphotomicrograph of a group of myofibers (traced in red), while thecorresponding GFP and Alkphos staining patterns are shown in the rightpanel. Photomicrographs of Alkphos staining were taken with a red filterto allow for superimposition of staining patterns with GFP fluorescence.Co-expression of Alkphos and GFP is shown within myofibers as ayellow/orange color. Myofibers are marked as follows: (−) negative forboth Alkphos and GFP, (*) positive for only GFP, and (+) positive forboth GFP and Alkphos.

FIG. 16. Rescue of circular intermediates and characterization of DNAhybridization patterns. Using the ampicillin resistance gene (amp) andbacterial ori incorporated into the AV.GFP3ori vector, the extent ofcircular intermediate formation was assessed by rescuing amp resistantplasmids following transformation of ⅕ the isolated Hirt DNA into E.coli Sure cells. Twenty plasmids from each muscle sample were preparedand analyzed by slot blot hybridization against GFP, Alkphos, and Amp³²P-labeled DNA probes. A representative group demonstrating thehybridization patterns is shown in Panel A. Panel B depicts the mean(+/−SEM) number of rescued bacterial plasmids that hybridized to eitherGPF alone, or to both GFP and Alkphos probes, following transformationof ⅕^(th) of the Hirt DNA. These numbers were calculated from thepercentage of plasmids hybridizing to GPF and/or Alkphos and the totalCFU plating efficiency derived from the original transformation. Intotal, 3 independent muscle samples were analyzed for a total of 60plasmids at each time point. The percentage of GFP hybridizationpositive rescued plasmids that also demonstrated hybridization toAlkphos is shown in Panel C. These data demonstrate an increase in theabundance of rescued GFP/Alkphos co-encoding circular intermediates overtime.

FIG. 17. Transgene expression from rescued circular intermediates.Rescued circular intermediate plasmids were transfected into 293 cellsfor assessment of their ability to express encoded transgenes. In thesestudies all GFP hybridization positive clones from at least two muscleswere tested for each time point and scored for their ability to expressGFP and Alkaline phosphatase. In total at least 40 clones were evaluatedfor each time point. Three patterns of transgene expression wereobserved following transfection of these plasmids: I) no gene expression(Panel A), II) GFP expression only (Panel B), and III) GFP and Alkphosexpression (Panel C). Panels A-C depict Nomarski photomicrographs (left)of GFP fluorescent fields (center) and Alkphos staining of a differentfield from the same culture (right). The percentage of GFP hybridizationpositive clones that also expressed GFP is shown in Panel D.Additionally, this panel illustrates the percentage of GFP expressingclones also expressing Alkphos.

FIG. 18. Structural analysis of bi-functional concatamer circularintermediates. To fully characterize the nature of GFP and Alkphosco-expressing circular intermediates, detailed structural analyses wereperformed using restriction enzyme mapping and Southern blothybridization with GFP, Alkphos, and ITR ³²P-labeled probes. Resultsfrom Southern blot analysis of plasmid clone #33 (Panel A) and clone #5(Panel C) are given as representative examples of circular intermediatesisolated from 80 and 35 day Hirt DNA of rAAV infected muscle,respectively. Agarose gels were run in triplicate for each of theseclones and Southern blot filters were hybridized with one of the threeDNA probes as indicated below each autoradiogram. Molecular weights (kb)are indicated to the left of the ethidium stained agarose gel andrestriction enzymes are marked on the top of each gel/filter. Panels Band D give the deduced structure of plasmid clones #33 and #5,respectively, as based on Southern blot analysis. For ease of comparisonwith the restriction maps of the viral genomes given in FIG. 14A, theposition of restriction enzyme sites (kb) are marked with the indicatedorientation of intact viral genomes. However, in clone #33 a deletionoccurred between the AseI and HindIII site of a head-to-tail arraybetween AV.Alkphos and AV.GFP3ori, as reflected by a 900 bp reduction inthe anticipated size of HindIII/NotI and ClaI/AseI fragments (marked byasterisks in Panel A). Furthermore, the SphI site flanking an ITR wasablated in clone #5 (bands effected by this deletion are marked byasterisks in Panel C). The deletion is not reflected in the overallconcatamer since the exact region involved and/or the size of thedeletion is unclear. Additionally, chemical sequence evidence of rescuedcircular intermediates suggests that the predominant form of ITR arraysmay be in a double-D structure (ie., one ITR flanked by two D-sequencerather than two ITRs) and hence ITR arrays containing fragments mayappear 147 bp shorter than indicated. However, to more easily depict theorientation of viral genomes, the position of 5′ and 3′ ITRs isindicated rather than representing a single ITR at these junctions.

FIG. 19. Application of rAAV circular concatamers to deliver vectorswith large gene inserts. Panel A depicts two rAAV vectors encoding twohalves of a cDNA (red) and flanked by splice site consensus sequences(brown). Panel B depicts one potential type of intermolecular concatamerfollowing co-infection of cells with the independent vectors shown inpanel A. Full length transgene mRNA can then be produced by splicingbetween these two vector encoded sequences within circular concatamers.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the terms “isolated and/or purified” refer to in vitropreparation, isolation and/or purification of a nucleic acid molecule ofthe invention, so that it is not associated with in vivo substances.

As used herein, a DNA molecule, sequence or segment of the inventionpreferably is biologically active. A biologically active DNA molecule ofthe invention has at least about 1%, more preferably at least about 10%,and more preferably at least about 50%, of the activity of a DNAmolecule comprising ITR sequences from a circular intermediate of AAV,e.g., a DNA molecule comprising SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,SEQ ID NO:6, or a subunit or variant thereof. The activity of a nucleicacid molecule of the invention can be measured by methods well known tothe art, some of which are described hereinbelow. For example, thepresence of the DNA molecule in a recombinant nucleic acid molecule in ahost cell results in episomal persistence and/or increased abundance ofthe recombinant molecule in those cells relative to corresponding cellshaving a recombinant nucleic acid molecule lacking a DNA molecule of theinvention.

A variant DNA molecule, sequence or segment of the invention has atleast about 70%, preferably at least about 80%, and more preferably atleast about 90%, but less than 100%, contiguous nucleotide sequencehomology or identity to a DNA molecule comprising ITR sequences from acircular intermediate of AAV, e.g., SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, a subunit thereof. A variant DNA molecule of theinvention may include nucleotide bases not present in SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, e.g., 5′, 3′ or internal deletions orinsertions, such as the insertion of a restriction endonucleaserecognition site, so long as these bases do not substantially reduce thebiological activity of the molecule. A substantial reduction in activitymeans a reduction in activity of greater than about 50%, preferablygreater than about 90%.

I. Identification of Nucleic Acid Molecules Falling within the Scope ofthe Invention

A. Nucleic Acid Molecules of the Invention 1. Sources of the NucleicAcid Molecules of the Invention

Sources of nucleotide sequences from which the present nucleic acidmolecules can be obtained include AAV infected cells, e.g., anyvertebrate, preferably mammalian, cellular source.

As used herein, the terms “isolated and/or purified” refer to in vitroisolation of a nucleic acid, e.g., DNA molecule from its naturalcellular environment, and from association with other components of thecell, such as nucleic acid or polypeptide, so that it can be sequenced,replicated, and/or expressed. For example, “isolated nucleic acid” isRNA or DNA containing greater than about 50, preferably about 300, andmore preferably about 500 or more, sequential nucleotide bases thatcomprise a DNA segment from a circular intermediate of AAV whichcontains at least a portion of the 5′ and 3′ ITRs and the D sequence, ora variant thereof, that is complementary or hybridizes, respectively, toAAV ITR DNA and remains stably bound under stringent conditions, asdefined by methods well known in the art, e.g., in Sambrook et al.,1989. Thus, the RNA or DNA is “isolated” in that it is free from atleast one contaminating nucleic acid with which it is normallyassociated in the natural source of the RNA or DNA and is preferablysubstantially free of any other mammalian RNA or DNA. The phrase “freefrom at least one contaminating source nucleic acid with which it isnormally associated” includes the case where the nucleic acid isreintroduced into the source or natural cell but is in a differentchromosomal location or is otherwise flanked by nucleic acid sequencesnot normally found in the source cell, e.g., in a vector or plasmid. Anexample of isolated nucleic acid within the scope of the invention isnucleic acid that shares at least about 80%, preferably at least about90%, and more preferably at least about 95%, sequence identity with SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, or a subunit thereof.

As used herein, the term “recombinant nucleic acid” or “preselectednucleic acid,” e.g., “recombinant DNA sequence or segment” or“preselected DNA sequence or segment” refers to a nucleic acid, e.g., toDNA, that has been derived or isolated from any appropriate cellularsource, that may be subsequently chemically altered in vitro, so thatits sequence is not naturally occurring, or corresponds to naturallyoccurring sequences that are not positioned as they would be positionedin a genome which has not been transformed with exogenous DNA. Anexample of preselected DNA “derived” from a source, would be a DNAsequence that is identified as a useful fragment within a givenorganism, and which is then chemically synthesized in essentially pureform. An example of such DNA “isolated” from a source would be a usefulDNA sequence that is excised or removed from said source by chemicalmeans, e.g., by the use of restriction endonucleases, so that it can befurther manipulated, e.g., amplified, for use in the invention, by themethodology of genetic engineering.

Thus, recovery or isolation of a given fragment of DNA from arestriction digest can employ separation of the digest on polyacrylamideor agarose gel by electrophoresis, identification of the fragment ofinterest by comparison of its mobility versus that of marker DNAfragments of known molecular weight, removal of the gel sectioncontaining the desired fragment, and separation of the gel from DNA. SeeLawn et al., Nucleic Acids Res., 9, 6103 (1981), and Goeddel et al.,Nucleic Acids Res., 8, 4057 (1980). Therefore, “preselected DNA”includes completely synthetic DNA sequences, semi-synthetic DNAsequences, DNA sequences isolated from biological sources, and DNAsequences derived from RNA, as well as mixtures thereof.

Nucleic acid molecules having base pair substitutions (i.e., variants)are prepared by a variety of methods known in the art. These methodsinclude, but are not limited to, isolation from a natural source (in thecase of naturally occurring sequence variants) or preparation byoligonucleotide-mediated (or site-directed) mutagenesis, PCRmutagenesis, and cassette mutagenesis of an earlier prepared variant ora non-variant version of the nucleic acid molecule.

Oligonucleotide-mediated mutagenesis is a preferred method for preparingsubstitution variants. This technique is well known in the art asdescribed by Adelman et al., DNA, 2, 183 (1983). Briefly, AAV DNA isaltered by hybridizing an oligonucleotide encoding the desired mutationto a DNA template, where the template is the single-stranded form of aplasmid or bacteriophage containing the unaltered or native DNA sequenceof AAV. After hybridization, a DNA polymerase is used to synthesize anentire second complementary strand of the template that will thusincorporate the oligonucleotide primer, and will code for the selectedalteration in the AAV DNA.

Generally, oligonucleotides of at least 25 nucleotides in length areused. An optimal oligonucleotide will have 12 to 15 nucleotides that arecompletely complementary to the template on either side of thenucleotide(s) coding for the mutation. This ensures that theoligonucleotide will hybridize properly to the single-stranded DNAtemplate molecule. The oligonucleotides are readily synthesized usingtechniques known in the art such as that described by Crea et al., Proc.Natl. Acad. Sci. U.S.A., 75, 5765 (1978).

The DNA template can be generated by those vectors that are eitherderived from bacteriophage M13 vectors (the commercially availableM13mp18 and M13mp19 vectors are suitable), or those vectors that containa single-stranded phage origin of replication as described by Viera etal., Meth. Enzymol., 153, 3 (1987). Thus, the DNA that is to be mutatedmay be inserted into one of these vectors to generate single-strandedtemplate. Production of the single-stranded template is described inSections 4.21-4.41 of Sambrook et al., Molecular Cloning: A LaboratoryManual (Cold Spring Harbor Laboratory Press, N.Y. 1989).

Alternatively, single-stranded DNA template may be generated bydenaturing double-stranded plasmid (or other) DNA using standardtechniques.

For alteration of the native DNA sequence (to generate amino acidsequence variants, for example), the oligonucleotide is hybridized tothe single-stranded template under suitable hybridization conditions. ADNA polymerizing enzyme, usually the Klenow fragment of DNA polymeraseI, is then added to synthesize the complementary strand of the templateusing the oligonucleotide as a primer for synthesis. A heteroduplexmolecule is thus formed such that one strand of DNA encodes the mutatedform of AAV, and the other strand (the original template) encodes thenative, unaltered sequence of AAV. This heteroduplex molecule is thentransformed into a suitable host cell, usually a prokaryote such as E.coli JM101. After the cells are grown, they are plated onto agaroseplates and screened using the oligonucleotide primer radiolabeled with32-phosphate to identify the bacterial colonies that contain the mutatedDNA. The mutated region is then removed and placed in an appropriatevector, generally an expression vector of the type typically employedfor transformation of an appropriate host.

The method described immediately above may be modified such that ahomoduplex molecule is created wherein both strands of the plasmidcontain the mutations(s). The modifications are as follows: Thesingle-stranded oligonucleotide is annealed to the single-strandedtemplate as described above. A mixture of three deoxyribonucleotides,deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), anddeoxyribothymidine (dTTP), is combined with a modifiedthiodeoxyribocytosine called dCTP-(αS) (which can be obtained from theAmersham Corporation). This mixture is added to thetemplate-oligonucleotide complex. Upon addition of DNA polymerase tothis mixture, a strand of DNA identical to the template except for themutated bases is generated. In addition, this new strand of DNA willcontain dCTP-(αS) instead of dCTP, which serves to protect it fromrestriction endonuclease digestion.

After the template strand of the double-stranded heteroduplex is nickedwith an appropriate restriction enzyme, the template strand can bedigested with ExoIII nuclease or another appropriate nuclease past theregion that contains the site(s) to be mutagenized. The reaction is thenstopped to leave a molecule that is only partially single-stranded. Acomplete double-stranded DNA homoduplex is then formed using DNApolymerase in the presence of all four deoxyribonucleotidetriphosphates, ATP, and DNA ligase. This homoduplex molecule can then betransformed into a suitable host cell such as E. coli JM101.

For example, a preferred embodiment of the invention is an isolated andpurified DNA molecule comprising a DNA segment comprising SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, a subunit thereof or a variantthereof having nucleotide substitutions, or deletions or insertions.

II. Preparation of Molecules Useful to Practice the Methods of theInvention A. Nucleic Acid Molecules 1. Chimeric Expression Cassettes

To prepare expression cassettes for transformation herein, therecombinant or preselected DNA sequence or segment may be circular orlinear, double-stranded or single-stranded. Generally, the preselectedDNA sequence or segment is in the form of chimeric DNA, such as plasmidDNA, that can also contain coding regions flanked by control sequenceswhich promote the expression of the preselected DNA present in theresultant cell line.

As used herein, “chimeric” means that a vector comprises DNA from atleast two different species, or comprises DNA from the same species,which is linked or associated in a manner which does not occur in the“native” or wild type of the species.

Aside from the preselected DNA sequences described above, a portion ofthe preselected DNA may serve a regulatory or a structural function. Forexample, the preselected DNA may itself comprise a promoter that isactive in mammalian cells, or may utilize a promoter already present inthe genome that is the transformation target. Such promoters include theCMV promoter, as well as the SV40 late promoter and retroviral LTRs(long terminal repeat elements), although many other promoter elementswell known to the art may be employed in the practice of the invention.

Other elements functional in the host cells, such as introns, enhancers,polyadenylation sequences and the like, may also be a part of thepreselected DNA. Such elements may or may not be necessary for thefunction of the DNA, but may provide improved expression of the DNA byaffecting transcription, stability of the mRNA, or the like. Suchelements may be included in the DNA as desired to obtain the optimalperformance of the transforming DNA in the cell.

“Control sequences” is defined to mean DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotic cells,for example, include a promoter, and optionally an operator sequence,and a ribosome binding site. Eukaryotic cells are known to utilizepromoters, polyadenylation signals, and enhancers.

“Operably linked” is defined to mean that the nucleic acids are placedin a functional relationship with another nucleic acid sequence. Forexample, DNA for a presequence or secretory leader is operably linked toDNA for a peptide or polypeptide if it is expressed as a preprotein thatparticipates in the secretion of the peptide or polypeptide; a promoteror enhancer is operably linked to a coding sequence if it affects thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to facilitatetranslation. Generally, “operably linked” means that the DNA sequencesbeing linked are contiguous and, in the case of a secretory leader,contiguous and in reading phase. However, enhancers do not have to becontiguous. Linking is accomplished by ligation at convenientrestriction sites. If such sites do not exist, the syntheticoligonucleotide adaptors or linkers are used in accord with conventionalpractice.

The preselected DNA to be introduced into the cells further willgenerally contain either a selectable marker gene or a reporter gene orboth to facilitate identification and selection of transformed cellsfrom the population of cells sought to be transformed. Alternatively,the selectable marker may be carried on a separate piece of DNA and usedin a co-transformation procedure. Both selectable markers and reportergenes may be flanked with appropriate regulatory sequences to enableexpression in the host cells. Useful selectable markers are well knownin the art and include, for example, antibiotic and herbicide-resistancegenes, such as neo, hpt, dhfr, bar, aroA, dapA and the like. See also,the genes listed on Table 1 of Lundquist et al. (U.S. Pat. No.5,848,956).

Reporter genes are used for identifying potentially transformed cellsand for evaluating the functionality of regulatory sequences. Reportergenes which encode for easily assayable proteins are well known in theart. In general, a reporter gene is a gene which is not present in orexpressed by the recipient organism or tissue and which encodes aprotein whose expression is manifested by some easily detectableproperty, e.g., enzymatic activity. Preferred genes include thechloramphenicol acetyl transferase gene (cat) from Tn9 of E. coli, thebeta-glucuronidase gene (gus) of the uidA locus of E. coli, and theluciferase gene from firefly Photinus pyralis. Expression of thereporter gene is assayed at a suitable time after the DNA has beenintroduced into the recipient cells.

The general methods for constructing recombinant DNA which can transformtarget cells are well known to those skilled in the art, and the samecompositions and methods of construction may be utilized to produce theDNA useful herein. For example, J. Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press (2d ed., 1989),provides suitable methods of construction.

2. Transformation into Host Cells

The recombinant DNA can be readily introduced into the host cells, e.g.,mammalian, bacterial, yeast or insect cells by transfection with anexpression vector of the invention, by any procedure useful for theintroduction into a particular cell, e.g., physical or biologicalmethods, to yield a transformed cell having the recombinant DNA stablyintegrated into its genome or present as an episome which can persist inthe transformed cells, so that the DNA molecules, sequences, orsegments, of the present invention are maintained and/or expressed bythe host cell.

Physical methods to introduce a preselected DNA into a host cell includecalcium phosphate precipitation, lipofection, particle bombardment,microinjection, electroporation, and the like. Biological methods tointroduce the DNA of interest into a host cell include the use of DNAand RNA viral vectors. The main advantage of physical methods is thatthey are not associated with pathological or oncogenic processes ofviruses. However, they are less precise, often resulting in multiplecopy insertions, random integration, disruption of foreign andendogenous gene sequences, and unpredictable expression.

As used herein, the term “cell line” or “host cell” is intended to referto well-characterized homogenous, biologically pure populations ofcells. These cells may be eukaryotic cells that are neoplastic or whichhave been “immortalized” in vitro by methods known in the art, as wellas primary cells, or prokaryotic cells. The cell line or host cell ispreferably of mammalian origin, but cell lines or host cells ofnon-mammalian origin may be employed, including plant, insect, yeast,fungal or bacterial sources. Generally, the preselected DNA sequence isrelated to a DNA sequence which is resident in the genome of the hostcell but is not expressed, or not highly expressed, or, alternatively,overexpressed.

“Transfected” or “transformed” is used herein to include any host cellor cell line, the genome of which has been altered or augmented by thepresence of at least one preselected DNA sequence, which DNA is alsoreferred to in the art of genetic engineering as “heterologous DNA,”“recombinant DNA,” “exogenous DNA,” “genetically engineered,”“non-native,” or “foreign DNA,” wherein said DNA was isolated andintroduced into the genome of the host cell or cell line by the processof genetic engineering. The host cells of the present invention aretypically produced by transfection with a DNA sequence in a plasmidexpression vector, a viral expression vector, or as an isolated linearDNA sequence.

To confirm the presence of the preselected DNA sequence in the hostcell, a variety of assays may be performed. Such assays include, forexample, “molecular biological” assays well known to those of skill inthe art, such as Southern and Northern blotting, RT-PCR and PCR;“biochemical” assays, such as detecting the presence of a polypeptideexpressed from a gene present in the vector, e.g., by immunologicalmeans (immunoprecipitations, immunoaffinity columns, ELISAs and Westernblots) or by any other assay useful to identify molecules falling withinthe scope of the invention.

To detect and quantitate RNA produced from introduced DNA segments,RT-PCR may be employed. In this application of PCR, it is firstnecessary to reverse transcribe RNA into DNA, using enzymes such asreverse transcriptase, and then through the use of conventional PCRtechniques amplify the DNA. In most instances PCR techniques, whileuseful, will not demonstrate integrity of the RNA product. Furtherinformation about the nature of the RNA product may be obtained byNorthern blotting. This technique demonstrates the presence of an RNAspecies and gives information about the integrity of that RNA. Thepresence or absence of an RNA species can also be determined using dotor slot blot Northern hybridizations. These techniques are modificationsof Northern blotting and only demonstrate the presence or absence of anRNA species.

While Southern blotting and PCR may be used to detect the DNA segment inquestion, they do not provide information as to whether the DNA segmentis being expressed. Expression may be evaluated by specificallyidentifying the polypeptide products of the introduced DNA sequences orevaluating the phenotypic changes brought about by the expression of theintroduced DNA segment in the host cell.

III. Dosages Formulations and Routes of Administration

Administration of a nucleic acid molecule may be accomplished throughthe introduction of cells transformed with the nucleic acid molecule(see, for example, WO 93/02556), the administration of the nucleic acidmolecule itself (see, for example, Felgner et al., U.S. Pat. No.5,580,859, Pardoll et al., Immunity, 3, 165 (1995); Stevenson et al.,Immunol. Rev., 145, 211 (1995); Molling, J. Mol. Med., 75, 242 (1997);Donnelly et al., Ann. N.Y. Acad. Sci., 772, 40 (1995); Yang et al., Mol.Med. Today, 2, 476 (1996); Abdallah et al., Biol. Cell, 85, 1 (1995)),through infection with a recombinant virus or via liposomes.Pharmaceutical formulations, dosages and routes of administration fornucleic acids are generally disclosed, for example, in Felgner et al.,supra.

Administration of the therapeutic agents in accordance with the presentinvention may be continuous or intermittent, depending, for example,upon the recipient's physiological condition, whether the purpose of theadministration is therapeutic or prophylactic, and other factors knownto skilled practitioners. The administration of the agents of theinvention may be essentially continuous over a preselected period oftime or may be in a series of spaced doses. Both local and systemicadministration is contemplated. When the molecules of the invention areemployed for prophylactic purposes, agents of the invention are amenableto chronic use, preferably by systemic administration.

One or more suitable unit dosage forms comprising the therapeutic agentsof the invention, which, as discussed below, may optionally beformulated for sustained release, can be administered by a variety ofroutes including oral, or parenteral, including by rectal, transdermal,subcutaneous, intravenous, intramuscular, intraperitoneal,intrathoracic, intrapulmonary and intranasal routes. The formulationsmay, where appropriate, be conveniently presented in discrete unitdosage forms and may be prepared by any of the methods well known topharmacy. Such methods may include the step of bringing into associationthe therapeutic agent with liquid carriers, solid matrices, semi-solidcarriers, finely divided solid carriers or combinations thereof, andthen, if necessary, introducing or shaping the product into the desireddelivery system.

When the therapeutic agents of the invention are prepared for oraladministration, they are preferably combined with a pharmaceuticallyacceptable carrier, diluent or excipient to form a pharmaceuticalformulation, or unit dosage form. The total active ingredients in suchformulations comprise from 0.1 to 99.9% by weight of the formulation. By“pharmaceutically acceptable” it is meant the carrier, diluent,excipient, and/or salt must be compatible with the other ingredients ofthe formulation, and not deleterious to the recipient thereof. Theactive ingredient for oral administration may be present as a powder oras granules; as a solution, a suspension or an emulsion; or inachievable base such as a synthetic resin for ingestion of the activeingredients from a chewing gum. The active ingredient may also bepresented as a bolus, electuary or paste.

Pharmaceutical formulations containing the therapeutic agents of theinvention can be prepared by procedures known in the art using wellknown and readily available ingredients. For example, the agent can beformulated with common excipients, diluents, or carriers, and formedinto tablets, capsules, suspensions, powders, and the like. Examples ofexcipients, diluents, and carriers that are suitable for suchformulations include the following fillers and extenders such as starch,sugars, mannitol, and silicic derivatives; binding agents such ascarboxymethyl cellulose, HPMC and other cellulose derivatives,alginates, gelatin, and polyvinyl-pyrrolidone; moisturizing agents suchas glycerol; disintegrating agents such as calcium carbonate and sodiumbicarbonate; agents for retarding dissolution such as paraffin;resorption accelerators such as quaternary ammonium compounds; surfaceactive agents such as cetyl alcohol, glycerol monostearate; adsorptivecarriers such as kaolin and bentonite; and lubricants such as talc,calcium and magnesium stearate, and solid polyethyl glycols.

For example, tablets or caplets containing the agents of the inventioncan include buffering agents such as calcium carbonate, magnesium oxideand magnesium carbonate. Caplets and tablets can also include inactiveingredients such as cellulose, pregelatinized starch, silicon dioxide,hydroxy propyl methyl cellulose, magnesium stearate, microcrystallinecellulose, starch, talc, titanium dioxide, benzoic acid, citric acid,corn starch, mineral oil, polypropylene glycol, sodium phosphate, andzinc stearate, and the like. Hard or soft gelatin capsules containing anagent of the invention can contain inactive ingredients such as gelatin,microcrystalline cellulose, sodium lauryl sulfate, starch, talc, andtitanium dioxide, and the like, as well as liquid vehicles such aspolyethylene glycols (PEGs) and vegetable oil. Moreover, enteric coatedcaplets or tablets of an agent of the invention are designed to resistdisintegration in the stomach and dissolve in the more neutral toalkaline environment of the duodenum.

The therapeutic agents of the invention can also be formulated aselixirs or solutions for convenient oral administration or as solutionsappropriate for parenteral administration, for instance byintramuscular, subcutaneous or intravenous routes.

The pharmaceutical formulations of the therapeutic agents of theinvention can also take the form of an aqueous or anhydrous solution ordispersion, or alternatively the form of an emulsion or suspension.

Thus, the therapeutic agent may be formulated for parenteraladministration (e.g., by injection, for example, bolus injection orcontinuous infusion) and may be presented in unit dose form in ampules,pre-filled syringes, small volume infusion containers or in multi-dosecontainers with an added preservative. The active ingredients may takesuch forms as suspensions, solutions, or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredients may be in powder form, obtained by aseptic isolation ofsterile solid or by lyophilization from solution, for constitution witha suitable vehicle, e.g., sterile, pyrogen-free water, before use.

These formulations can contain pharmaceutically acceptable vehicles andadjuvants which are well known in the prior art. It is possible, forexample, to prepare solutions using one or more organic solvent(s) thatis/are acceptable from the physiological standpoint, chosen, in additionto water, from solvents such as acetone, ethanol, isopropyl alcohol,glycol ethers such as the products sold under the name “Dowanol”,polyglycols and polyethylene glycols, C₁-C₄ alkyl esters of short-chainacids, preferably ethyl or isopropyl lactate, fatty acid triglyceridessuch as the products marketed under the name “Miglyol”, isopropylmyristate, animal, mineral and vegetable oils and polysiloxanes.

The compositions according to the invention can also contain thickeningagents such as cellulose and/or cellulose derivatives. They can alsocontain gums such as xanthan, guar or carbo gum or gum arabic, oralternatively polyethylene glycols, bentones and montmorillonites, andthe like.

It is possible to add, if necessary, an adjuvant chosen fromantioxidants, surfactants, other preservatives, film-forming,keratolytic or comedolytic agents, perfumes and colorings. Also, otheractive ingredients may be added, whether for the conditions described orsome other condition.

For example, among antioxidants, t-butylhydroquinone, butylatedhydroxyanisole, butylated hydroxytoluene and α-tocopherol and itsderivatives may be mentioned. The galenical forms chiefly conditionedfor topical application take the form of creams, milks, gels, dispersionor microemulsions, lotions thickened to a greater or lesser extent,impregnated pads, ointments or sticks, or alternatively the form ofaerosol formulations in spray or foam form or alternatively in the formof a cake of soap.

Additionally, the agents are well suited to formulation as sustainedrelease dosage forms and the like. The formulations can be soconstituted that they release the active ingredient only or preferablyin a particular part of the intestinal or respiratory tract, possiblyover a period of time. The coatings, envelopes, and protective matricesmay be made, for example, from polymeric substances, such aspolylactide-glycolates, liposomes, microemulsions, microparticles,nanoparticles, or waxes. These coatings, envelopes, and protectivematrices are useful to coat indwelling devices, e.g., stents, catheters,peritoneal dialysis tubing, and the like.

The therapeutic agents of the invention can be delivered via patches fortransdermal administration. See U.S. Pat. No. 5,560,922 for examples ofpatches suitable for transdermal delivery of a therapeutic agent.Patches for transdermal delivery can comprise a backing layer and apolymer matrix which has dispersed or dissolved therein a therapeuticagent, along with one or more skin permeation enhancers. The backinglayer can be made of any suitable material which is impermeable to thetherapeutic agent. The backing layer serves as a protective cover forthe matrix layer and provides also a support function. The backing canbe formed so that it is essentially the same size layer as the polymermatrix or it can be of larger dimension so that it can extend beyond theside of the polymer matrix or overlay the side or sides of the polymermatrix and then can extend outwardly in a manner that the surface of theextension of the backing layer can be the base for an adhesive means.Alternatively, the polymer matrix can contain, or be formulated of, anadhesive polymer, such as polyacrylate or acrylate/vinyl acetatecopolymer. For long-term applications it might be desirable to usemicroporous and/or breathable backing laminates, so hydration ormaceration of the skin can be minimized.

Examples of materials suitable for making the backing layer are films ofhigh and low density polyethylene, polypropylene, polyurethane,polyvinylchloride, poly-esters such as poly(ethylene phthalate), metalfoils, metal foil laminates of such suitable polymer films, and thelike. Preferably, the materials used for the backing layer are laminatesof such polymer films with a metal foil such as aluminum foil. In suchlaminates, a polymer film of the laminate will usually be in contactwith the adhesive polymer matrix.

The backing layer can be any appropriate thickness which will providethe desired protective and support functions. A suitable thickness willbe from about 10 to about 200 microns.

Generally, those polymers used to form the biologically acceptableadhesive polymer layer are those capable of forming shaped bodies, thinwalls or coatings through which therapeutic agents can pass at acontrolled rate. Suitable polymers are biologically and pharmaceuticallycompatible, nonallergenic and insoluble in and compatible with bodyfluids or tissues with which the device is contacted. The use of solublepolymers is to be avoided since dissolution or erosion of the matrix byskin moisture would affect the release rate of the therapeutic agents aswell as the capability of the dosage unit to remain in place forconvenience of removal.

Exemplary materials for fabricating the adhesive polymer layer includepolyethylene, polypropylene, polyurethane, ethylene/propylenecopolymers, ethylene/ethylacrylate copolymers, ethylene/vinyl acetatecopolymers, silicone elastomers, especially the medical-gradepolydimethylsiloxanes, neoprene rubber, polyisobutylene, polyacrylates,chlorinated polyethylene, polyvinyl chloride, vinyl chloride-vinylacetate copolymer, crosslinked polymethacrylate polymers (hydro-gel),polyvinylidene chloride, poly(ethylene terephthalate), butyl rubber,epichlorohydrin rubbers, ethylenevinyl alcohol copolymers,ethylene-vinyloxyethanol copolymers; silicone copolymers, for example,polysiloxane-polycarbonate copolymers, polysiloxanepolyethylene oxidecopolymers, polysiloxane-polymethacrylate copolymers,polysiloxane-alkylene copolymers (e.g., polysiloxane-ethylenecopolymers), polysiloxane-alkylenesilane copolymers (e.g.,polysiloxane-ethylenesilane copolymers), and the like; cellulosepolymers, for example methyl or ethyl cellulose, hydroxy propyl methylcellulose, and cellulose esters; polycarbonates;polytetrafluoroethylene; and the like.

Preferably, a biologically acceptable adhesive polymer matrix should beselected from polymers with glass transition temperatures below roomtemperature. The polymer may, but need not necessarily, have a degree ofcrystallinity at room temperature. Cross-linking monomeric units orsites can be incorporated into such polymers. For example, cross-linkingmonomers can be incorporated into polyacrylate polymers, which providesites for cross-linking the matrix after dispersing the therapeuticagent into the polymer. Known cross-linking monomers for polyacrylatepolymers include polymethacrylic esters of polyols such as butylenediacrylate and dimethacrylate, trimethylol propane trimethacrylate andthe like. Other monomers which provide such sites include allylacrylate, allyl methacrylate, diallyl maleate and the like.

Preferably, a plasticizer and/or humectant is dispersed within theadhesive polymer matrix. Water-soluble polyols are generally suitablefor this purpose. Incorporation of a humectant in the formulation allowsthe dosage unit to absorb moisture on the surface of skin which in turnhelps to reduce skin irritation and to prevent the adhesive polymerlayer of the delivery system from failing.

Therapeutic agents released from a transdermal delivery system must becapable of penetrating each layer of skin. In order to increase the rateof permeation of a therapeutic agent, a transdermal drug delivery systemmust be able in particular to increase the permeability of the outermostlayer of skin, the stratum corneum, which provides the most resistanceto the penetration of molecules. The fabrication of patches fortransdermal delivery of therapeutic agents is well known to the art.

For administration to the upper (nasal) or lower respiratory tract byinhalation, the therapeutic agents of the invention are convenientlydelivered from an insufflator, nebulizer or a pressurized pack or otherconvenient means of delivering an aerosol spray. Pressurized packs maycomprise a suitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, thecomposition may take the form of a dry powder, for example, a powder mixof the therapeutic agent and a suitable powder base such as lactose orstarch. The powder composition may be presented in unit dosage form in,for example, capsules or cartridges, or, e.g., gelatine or blister packsfrom which the powder may be administered with the aid of an inhalator,insufflator or a metered-dose inhaler.

For intra-nasal administration, the therapeutic agent may beadministered via nose drops, a liquid spray, such as via a plasticbottle atomizer or metered-dose inhaler. Typical of atomizers are theMistometer (Wintrop) and the Medihaler (Riker).

The local delivery of the therapeutic agents of the invention can alsobe by a variety of techniques which administer the agent at or near thesite of disease. Examples of site-specific or targeted local deliverytechniques are not intended to be limiting but to be illustrative of thetechniques available. Examples include local delivery catheters, such asan infusion or indwelling catheter, e.g., a needle infusion catheter,shunts and stents or other implantable devices, site specific carriers,direct injection, or direct applications.

For topical administration, the therapeutic agents may be formulated asis known in the art for direct application to a target area.Conventional forms for this purpose include wound dressings, coatedbandages or other polymer coverings, ointments, creams, lotions, pastes,jellies, sprays, and aerosols. Ointments and creams may, for example, beformulated with an aqueous or oily base with the addition of suitablethickening and/or gelling agents. Lotions may be formulated with anaqueous or oily base and will in general also contain one or moreemulsifying agents, stabilizing agents, dispersing agents, suspendingagents, thickening agents, or coloring agents. The active ingredientscan also be delivered via iontophoresis, e.g., as disclosed in U.S. Pat.No. 4,140,122; 4,383,529; or 4,051,842. The percent by weight of atherapeutic agent of the invention present in a topical formulation willdepend on various factors, but generally will be from 0.01% to 95% ofthe total weight of the formulation, and typically 0.1-25% by weight.

Drops, such as eye drops or nose drops, may be formulated with anaqueous or non-aqueous base also comprising one or more dispersingagents, solubilizing agents or suspending agents. Liquid sprays areconveniently delivered from pressurized packs. Drops can be deliveredvia a simple eye dropper-capped bottle, or via a plastic bottle adaptedto deliver liquid contents dropwise, via a specially shaped closure.

The therapeutic agent may further be formulated for topicaladministration in the mouth or throat. For example, the activeingredients may be formulated as a lozenge further comprising a flavoredbase, usually sucrose and acacia or tragacanth; pastilles comprising thecomposition in an inert base such as gelatin and glycerin or sucrose andacacia; and mouthwashes comprising the composition of the presentinvention in a suitable liquid carrier.

The formulations and compositions described herein may also containother ingredients such as antimicrobial agents, or preservatives.Furthermore, the active ingredients may also be used in combination withother therapeutic agents, for example, bronchodilators.

In particular, for delivery of a vector of the invention to a tissuesuch as muscle, any physical or biological method that will introducethe vector into the muscle tissue of a host animal can be employed.Vector means both a bare recombinant vector and vector DNA packaged intoviral coat proteins, as is well known for AAV administration. Simplydissolving an AAV vector in phosphate buffered saline has beendemonstrated to be sufficient to provide a vehicle useful for muscletissue expression, and there are no known restrictions on the carriersor other components that can be coadministered with the vector (althoughcompositions that degrade DNA should be avoided in the normal mannerwith vectors). Pharmaceutical compositions can be prepared as injectableformulations or as topical formulations to be delivered to the musclesby transdermal transport. Numerous formulations for both intramuscularinjection and transdermal transport have been previously developed andcan be used in the practice of the invention. The vectors can be usedwith any pharmaceutically acceptable carrier for ease of administrationand handling.

For purposes of intramuscular injection, solutions in an adjuvant suchas sesame or peanut oil or in aqueous propylene glycol can be employed,as well as sterile aqueous solutions. Such aqueous solutions can bebuffered, if desired, and the liquid diluent first rendered isotonicwith saline or glucose. Solutions of the AAV vector as a free acid (DNAcontains acidic phosphate groups) or a pharmacologically acceptable saltcan be prepared in water suitably mixed with a surfactant such ashydroxypropylcellulose. A dispersion of AAV viral particles can also beprepared in glycerol, liquid polyethylene glycols and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. In this connection, the sterile aqueous media employedare all readily obtainable by standard techniques well-known to thoseskilled in the art.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol and the like), suitable mixtures thereof, andvegetable oils. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of a dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal andthe like. In many cases it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by use of agentsdelaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the AAVvector in the required amount in the appropriate solvent with various ofthe other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the sterilized active ingredient into a sterile vehiclewhich contains the basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and the freeze drying techniquewhich yield a powder of the active ingredient plus any additionaldesired ingredient from the previously sterile-filtered solutionthereof.

For purposes of topical administration, dilute sterile, aqueoussolutions (usually in about 0.1% to 5% concentration), otherwise similarto the above parenteral solutions, are prepared in containers suitablefor incorporation into a transdermal patch, and can include knowncarriers, such as pharmaceutical grade dimethylsulfoxide (DMSO).

The therapeutic compounds of this invention may be administered to amammal alone or in combination with pharmaceutically acceptablecarriers. As noted above, the relative proportions of active ingredientand carrier are determined by the solubility and chemical nature of thecompound, chosen route of administration and standard pharmaceuticalpractice.

The dosage of the present therapeutic agents which will be most suitablefor prophylaxis or treatment will vary with the form of administration,the particular compound chosen and the physiological characteristics ofthe particular patient under treatment. Generally, small dosages will beused initially and, if necessary, will be increased by small incrementsuntil the optimum effect under the circumstances is reached. Exemplarydosages are set out in the example below.

Since AAV has been shown to have a broad host range (for pulmonaryexpression) and persists in muscle, the vectors of the invention may beemployed to express a gene in any animal, and particularly in mammals,birds, fish, and reptiles, especially domesticated mammals and birdssuch as cattle, sheep, pigs, horses, dogs, cats, chickens, and turkeys.Both human and veterinary uses are particularly preferred.

The gene being expressed can be either a DNA segment encoding a protein,with whatever control elements (e.g., promoters, operators) are desiredby the user, or a non-coding DNA segment, the transcription of whichproduces all or part of some RNA-containing molecule (such as atranscription control element, +RNA, or anti-sense molecule).

Muscle tissue is a very attractive target for in vivo gene delivery andgene therapy, because it is not a vital organ and is very easy toaccess. If a disease is caused by a defective gene product which isrequired to be produced and/or secreted, such as hemophilia, diabetesand Gaucher's disease, and the like, is muscle is a good candidate tosupply the gene product if the appropriate gene can be effectivelydelivered into the cells.

Different vectors, such as naked DNA, adenovirus and retrovirus, havebeen utilized to directly deliver various transgenes into muscletissues. However, neither system can offer both high efficiency andlong-term expression. For naked plasmid DNA directly delivered intomuscle tissue, the efficiency is not high. There are only a few cellsnear the injection site that can maintain transgene expression.Furthermore, the plasmid DNA in the cells remains as non-replicatingepisomes, i.e., in the unintegrated form. Therefore, it will beeventually lost. For adenovirus vector, it can infect the non-dividingcells, and therefore, can be directly delivered into the mature tissuessuch as muscle. However, the transgene delivered by adenovirus vectorsare not useful to maintain long-term expression for the followingreasons. First, since adenovirus vectors still retain most of the viralgenes, they are not very safe. Moreover, the expression of those genescan cause the immune system to destroy the cells containing the vectors(see, for example, Yang et al. 1994, Proc. Natl. Acad. Sci.91:4407-4411). Second, since adenovirus is not an integration virus, itsDNA will eventually be diluted or degraded in the cells. Third, due tothe immune response, adenovirus vector could not be repeatedlydelivered. In the case of lifetime diseases, this will be a majorlimitation. For retrovirus vectors, although they can achieve stableintegration into the host chromosomes, their use is very restrictedbecause they can only infect dividing cells while a large majority ofthe muscle cells are non-dividing.

Adeno-associated virus vectors have certain advantages over theabove-mentioned vector systems. First, like adenovirus, AAV canefficiently infect non-dividing cells. Second, all the AAV viral genesare eliminated in the vector. Since the viral-gene-expression-inducedimmune reaction is no longer a concern, AAV vectors are safer than Advectors. Thirds, AAV is an integration virus by nature, and integrationinto the host chromosome will stably maintain its transgene in thecells. Fourth, AAV is an extremely stable virus, which is resistant tomany detergents, pH changes and heat (stable at 56° C. for more than anhour). It can be lyophilized and redissolved without losing itsactivity. Therefore, it is a very promising delivery vehicle for genetherapy.

The invention will be further described by, but is not limited to, thefollowing examples.

Example 1 Materials and Methods

Construction of rAAV Shuttle Vector.

A recombinant AAV shuttle vector (AV.GFP3ori) which contained a GFPtransgene cassette, bacterial ampicillin resistance gene, and bacterialorigin of replication, was generated from a cis-acting plasmid(pCisAV.GFP3ori). Expression of the GFP gene was directed by the CMVpromoter/enhancer and SV40 poly-adenylation sequences. pCisAV.GFP3oriwas constructed with pSub201 derived ITR elements (Samulski et al.,1987) and the intactness of ITR sequences was confirmed by restrictionanalysis with SmaI and PvuII, and by sequencing. Recombinant AAV stockswere generated by co-transfection of pCisAV.GFP3ori and pRep/Captogether with co-infection of recombinant Ad.CMVlacZ in 293 cells (Duanet al., 1997). Following transfection of forty 150 mm plates, cells werecollected at 72 hours by centrifugation and resuspended in 12 ml ofbuffer (10 mM Tris pH 8.0). Virus was released from cells by threecycles of freeze/thawing and passaged through a 25 gauge needle sixtimes. Cell lysates were then treated with 1.3 mg/ml DNase I at 37° C.for 30 minutes and 1% deoxycholate (g/ml final) and 0.05% trypsin (g/mlfinal) at 37° C. for 30 minutes. Samples were then placed on ice for 10minutes and centrifuged to remove large particulate material at 3,000rpm for 30 minutes.

rAAV was purified by isopycnic density gradient centrifugation in CsCl(r=1.4) in a SW55 rotor for 72 hours at 35K. Peak fractions of AAV werecombined and re-purified through two more rounds of CsCl centrifugation,followed by heating at 58° C. for 60 minutes to inactivate allcontaminant helper adenovirus. Typically, this preparation gaveapproximate AAV titers of 10¹² DNA molecules/ml and 2.5×10⁸GFP-expressing units/ml. Recombinant viral titers were assessed by slotblot and quantified against pCisAV.GFP3ori controls for DNA particles.Functional transducing units were quantified by GFP transgene expressionin 293 cells. The absence of helper adenovirus was confirmed byhistochemical staining of rAAV infected 293 cells forbeta-galactosidase, and no recombinant adenovirus was found in 10¹⁰particles of purified rAAV stocks. The absence of significant wtAAVcontamination was confirmed by immunocytochemical staining of rAAV/Adco-infected 293 cells with anti-Rep antibodies. These studies, which hada sensitivity of 1 wtAAV in 10¹⁰ rAAV particles, demonstrated an absenceof Rep staining as compared to pRep/Cap plasmid transfected controls.

Isolation and Structural Evaluation of AAV Circular Intermediates fromHela Cells.

Hela cells were grown in 35 mm dishes in DMEM media supplemented with10% fetal calf serum (FCS). Cells were infected in the presence of 2%FCS at 80% confluency with recombinant AV.GFP3ori (MOI=1000particles/cell, 1×10⁹ total particles/plate) and Hirt DNAs isolated asdescribed by Duan et al. (1997) at 6, 12, 24, 48, and 72 hourspost-infection. In experiments analyzing the effects of adenovirus,plates were co-infected with Ad.CMVLacZ (MOI=5000 particles/cell) in thepresence of 2% FCS/DMEM. Zero hour controls were generated by mixing 10⁹particles of AV.GFP3ori with cell lysates prior to Hirt DNA preparation.Hirt DNA isolated at each time point was used to transform E. coli SUREcells (Stratagene, La Jolla, Calif.). Typically, 1/10 of the Hirt DNApreparation was used to transform 40 ml of competent bacteria byelectroporation. The resultant total number of bacterial colonies wasquantified for each time point and the structure of circularintermediates was evaluated for greater than 20 plasmid clones for eachtime point from two independent experiments. Structural determinationswere based on restriction enzyme analysis using PstI, SphI, AseI singleand double digests together with Southern blotting against GFP, stuffer,and ITR probes.

Evaluation of E2a and GFP Gene Expression in Hela Cells.

E2a gene expression was evaluated by immunofluorescent staining of Helacells superinfected with E1-deleted Ad.CMVlacZ (MOI=0, 500, 5000particles/cell). Briefly, cells were fixed in methanol at −20° C. for 10minutes followed by air drying. Cells were then incubated at roomtemperature with hybridoma supernatant against Ad572 kd DBP (Reich etal., 1983), followed by goat anti-mouse-FITC antibody (5 mg/ml) for 30minutes at room temperature. In studies evaluating augmentation of AAVGFP transgene expression by adenovirus, Hela cells were harvested at 24or 72 hours post-infection by trypsinization, resuspended in 2% FCS/PBSand evaluated by FACS analyses. Thresholds were set using uninfectedcontrols and the percentage and/or the average relative fluorescentintensity was determined by sorting greater than 10⁵ cells perexperiment condition.

Sequence Analysis of AAV Circular Intermediates.

Sequence analysis of the ITR array within circular intermediates wasperformed using primers EL118 (5′-CGGGGGTCGTTGGGCGGTCA-3′; SEQ ID NO:1)and EL230 (5′-GGGCGGAGCCTATGGAAAA-3′; SEQ ID NO:2) which are nested to5′ and 3′ ITR sequences, respectively. Both circular and linearized(with SmaI which cuts within ITR sequences) plasmids were sequenced.

Results

Construction of rAAV Shuttle Vector and Isolation of CircularIntermediates.

To circumvent the inability to retrieve pre-integration intermediates oras stable episomal forms resistant to nuclease digestion, an alternativestrategy was developed to “trap” circular intermediates using arecombinant AAV shuttle vector. Recombinant AV.GFP3ori virus (FIG. 1B)was generated from a cis-acting plasmid (pCisAV.GFP3ori, FIG. 1A) byco-transfection in 293 cells with trans-acting plasmids encoding Rep andCap viral genes. This viral vector (AV.GFP3ori) encoded the greenfluorescent protein (GFP) reporter gene, a bacterial origin ofreplication (ori), and the bacterial ampicillin-resistance gene. Ori andampicillin-resistance sequences encoded in this virus allow for therescue of circular AAV genomes formed during the transduction process.

To test this strategy, Hela cells were infected with AV.GFP3ori(MOI=1000 particles/cell) and the abundance of circular intermediateswas evaluated following transformation of low molecular weight cellularHirt DNA into E. coli SURE cells. The presence of circular intermediateswas inferred by retrievable ampicillin-resistant bacterial colonies.Structural features of circular intermediates were determined byrestriction enzyme analysis and Southern blotting with various regionsof the provirus, including GFP, Stuffer, and ITR sequences.

The predominant circular form isolated after transduction of Hela cellswith AV.GFP3ori consisted of 4.7 kb monomer-sized molecules (FIG. 1C).SphI digestions of these circular intermediates yielded characteristic300 bp bands which hybridized to an ITR probe on Southern blots (FIG.2A). PstI, SphI, AseI single and double digests together with Southernblot analysis using GFP, Stuffer (data not shown), and ITR (FIG. 2A)probes confirmed the structure of the circular intermediates ashead-to-tail monomer genomes (FIG. 1C). In particular, PstI digeststogether with ITR Southern blots distinguish these head-to-tail circularintermediates from head-to-head or tail-to-tail circular dimers. Similarresults obtained with studies on AV.GFP3ori infected 293 cells andprimary fibroblasts have confirmed that monomer head-to-tail circularintermediates were also the most abundant form in these cell types.

Because the predicted molecular weight of an intact head-to-tail ITRSphI fragment would be approximately 360 bp, an anomalous migration inagarose gels might be due to the high secondary structure of invertedrepeats within ITRs. To this end, the head-to-tail orientation of theITRs, as predicted by Southern blot analysis, was confirmed usingseveral sequencing strategies. First, the SphI ITR hybridizing fragmentof a circular intermediates was subcloned into a secondary plasmidvector and sequenced with primers outside the ITR cloned sequences.These findings confirmed the head-to-tail orientation of ITRs.Additionally, sequence was obtained directly from six monomer circularintermediate clones using primers internal to both the 5′ and 3′ ITRs(FIG. 2C). In these studies, circular intermediates were digested withSmaI and the linear 4.6 kb plasmid was gel isolated prior to sequencing.SmaI digestion (which relaxed the secondary structure of ITRs) wasnecessary to obtain sequence information within the ITRs. The sequencingresults presented in FIG. 2C confirmed the orientation of head-to-tailITR arrays in these intermediates.

Interestingly, sequencing also revealed several consistent base pair(bp) changes in four of the six clones analyzed (FIG. 2C). These fourclones (p79, p81, p87, and p88) had consistent two bp changes within theD-sequence [G->A (122 bp) and A->G (125 bp)], which always occurredtogether with the bp alterations in the p5 promoter [A->G (114 bp) andA->C (115 bp)]. No other consistent bp changes were noted except for twoclones (p79 and p88) which demonstrated mutations just outside the 3′ITRD-sequence [T->G (381 bp) and T->C (383 bp)].

Although head-to-tail circular intermediates were the most abundantforms present in Hirt DNA from rAAV infected Hela cells, several lessfrequent structures were also detected. These included monomercircularized AAV genomes with one (p190) and three ITRs (p345) arrangedin a head-to-tail fashion as well as several clones with an unknownstructure lacking complete TTRs (p340) (FIG. 2A). Such diversity withinthe ITR array may represent homologous recombination in vivo or inbacteria during amplification. However, previous studies demonstratingsimilar variations in ITR sequences of head-to-tail integrated genomes,suggest that such changes in the length of the ITR array may occur invivo (Duan et al., 1997) Additionally, less frequent head-to-tailcircularized multimer forms were predicted based on the variation inmigration patterns of uncut plasmids which gave identical restrictionpatterns. Results shown in FIG. 2B confirmed the existence of monomerand dimer head-to-tail circular intermediates using partial digestionwith an enzyme which cuts once in the AAV genome (AseI). Cumulativeanalysis of greater than 200 independently isolated circularintermediates from Hela cells demonstrated that head-to-tail circularAAV genomes occurred in greatest abundance as monomers (92%) and lessfrequently as multimers of greater than one genome (8%).

To establish that head-to-tail circular intermediates were formed invivo and not by non-specific bacterial recombination of linear AAVgenomes present in the Hirt DNA, a set of reconstitution experiments wasperformed by which the same number of rAAV particles used for infectionexperiments were spiked into Hela cell lysates prior to Hirtpreparations. In these studies, background bacterial amplification ofHirt DNA spiked with linear rAAV genomes was negligible (FIG. 3D) and ofthe few isolated colonies obtained from these controls, none had apredicted head-to-tail structure as assessed by Southern blotrestriction enzyme analysis (FIG. 3E). Additionally, reconstitutionexperiments transforming bacterial with linearized dsDNA AAV genomes didnot give rise to significant levels of replication competent plasmids orthe characteristic head-to-tail structure associated with AAV circularintermediates. These findings confirm that circular intermediates do notlikely arise from non-specific recombination or ligation events witheither ssDNA or dsDNA linear AAV genomes in bacteria. Additional controlexperiments, demonstrating the lack of stuffer hybridizing sequences inAAV circular intermediates by Southern blotting, also confirm that thesestructures do not arise from contamination of viral stocks withpCisAV.GFP3ori plasmid.

The Formation of Head-to-Tail Circular AAV Intermediates is Augmented bySuperinfection with E1-Deleted Adenovirus.

Many aspects of the wtAAV growth cycle are affected by helperadenovirus, including AAV DNA replication, transcription, splicing,translation, and virion assembly. Such studies have provided concreteevidence that a subset of Ad early gene products provide helperfunctions for the wtAAV lytic cycle, including: E1a, E1b, E2a, E4 ORF6and VA1 RNA (Muzyczka, 1992). In this regard, one of the most criticalfactors which is required for AAV replication is the 34 kD E4 protein(ORF6). Recent observations on the helper function of Ad in rAAVtransduction have also demonstrated that Ad E4 ORF6 is essential for theaugmentation of rAAV transgene expression seen with adenovirusco-infection (Ferrari et al., 1996; Fisher et al., 1996). According tothese reports, the rate-limiting step enhanced by these adenoviralproteins is the conversion of single stranded AAV genomes to doublestranded forms.

Studies evaluating the kinetics of rAAV circular intermediate formationdemonstrated a time-dependent increase in abundance which peaked at 24hours post-infection in Hela cells and coincided with the onset of GFPtransgene expression (FIG. 3). To better understand the cellularmechanisms associated with AAV circular intermediate formation, theeffects of adenoviral co-infection on this process were evaluated. Theextent of transgene expression and circular intermediate formation inAV.GFP3ori infected Hela cells with or without co-infection withE1-deleted recombinant adenovirus was compared.

Although E1-deleted adenoviruses are severely handicapped in theirability to synthesize viral gene products, at high MOIs of >5000significant E2a protein expression was noted (FIG. 3A). As an indicatorof transgene expression, the abundance and average relative intensity ofGFP positive cells was determined against mock infected controls byfluorescent microscopy (FIG. 3B) and FACS analysis (FIG. 3C) at 72 hourspost-infection. In accord with previous reports demonstratingaugmentation in rAAV transgene expression by adenovirus (Ferrari et al.,1996; Fisher et al., 1996), the extent of GFP transgene expression wasdramatically increased at doses of adenovirus which led to viral geneexpression (MOI>5000; FIGS. 3A-C). Additionally, persistence of rAAVtransgene expression was also augmented by co-infection with E1-deletedadenovirus, as determined by GFP-expressing colony formation followingserial passages (FIG. 3C).

If circular intermediates represent a molecular form of rAAV importantfor efficient and/or persistent transgene expression, augmentation ofrAAV transgene expression by adenovirus might also modulate circularintermediate formation. In these studies, the abundance and time courseof AAV circular intermediate formation was evaluated followingsuperinfection with Ad.CMVLacZ. Results from these experiments are shownin FIG. 3D, which represents the total number of bacterial colonies (per35 mm plate) obtained following transformation of E. coli with Hirt DNAisolated from Hela cells infected with AV.GFP3ori (1000 DNAparticles/cell) with or without co-infection with Ad.CMVlacZ (5,000particles/cell). An MOI of 5000 Ad particles/cell was chosen for theseexperiments since this level of adenovirus led to minimal cytopathiceffect (CPE) with high levels of E2a expression.

These studies demonstrated a nearly 2-fold augmentation by Ad.CMVLacZ inthe total abundance of AAV rescued plasmid intermediates in E. coli(FIG. 3D). Southern blot restriction enzyme analysis demonstrated thatthe predominant forms in both the presence and absence of adenoviruswere head-to-tail monomer circular intermediates containing thediagnostic 300 bp ITR fragment following SphI digestion (FIG. 3E).Additionally, results demonstrated that adenovirus co-infection led toan earlier time of onset and increased stability of AAV head-to-tailmonomer circular intermediates (FIGS. 3E and F). For example, at 6 hourspost-infection, head-to-tail circular intermediates were only present inHela cells co-infected with adenovirus. Furthermore, a decline in thepercentage of head-to-tail circular intermediate clones was seen at48-72 hours post-AAV infection in the absence of adenovirus. Incontrast, this decline was significantly blunted by the presence ofhelper adenovirus (FIG. 3F). Based on these findings, it was concludedthat certain adenoviral proteins produced by superinfection withE1-deleted adenovirus were capable of modulating circular intermediatesformation and stability during rAAV transduction.

Discussion

In the present study, it was shown that circularization of linear AAVgenomes occurs during rAAV transduction. Circularization appears topredominately occur as head-to-tail monomer genomes. However, theexistence of less abundant circular multimer forms suggests thatrecombinational events subsequent to the initial infection may driveconcatamerization of circular genomes. The diversity in the length ofITR arrays found within circular intermediates (i.e., 1-3 ITRs) alsosupports the notion that these forms may be highly recombinagenic. Ofmechanistic interest in the formation of circular intermediates is theuniformity of mutations observed in the D-sequences and nearby p5promoter region and the confinement of these mutations to the 5′-ITRs.Although the etiology of these base pair changes is unknown, theiruniformity suggests that they may have a direct role in the formation ofcircular intermediates and in increased stability. Recent findings,which suggest that an endogenous host single strand D-sequence bindingprotein is important in rAAV transduction, lend support to the potentialinvolvement of this sequence in circular intermediate formation (Wang etal., 1997; Qing et al., 1998). Furthermore, it remains to be determinedwhether the in vivo formation of AAV circular intermediates occursthrough the circularization of single or double stranded AAV genomes.

By analogy, retroviral transduction intermediates have strikingsimilarities to the current findings with AAV. Three DNA forms have beenisolated following retroviral infection, including linear DNA with longterminal repeats (LTRs) at both ends, circular DNA with one LTR, andcircular DNA with multiple LTRs (Panganiban, 1985). Although it isdisputed which of these forms are the direct precursor to integration,the existence of circular retroviral genomes which also have similarrepeat regions at the ends of their genomes suggests the potential forcommon mechanisms with the formation of AAV circular intermediates.These AAV circular intermediates could act as integration precursorsand/or stable episomal genomes.

The head-to-tail ITR structures found in AAV circular intermediates aremost characteristic of latent integrated AAV genomes. In contrast, lyticphases of AAV growth are typically associated with head-to-head andtail-to-tail replication form genomes. Hence, it is likely that circularintermediates represent a latent aspect of the AAV life cycle. Thefinding that co-infection with adenovirus leads to increased abundanceand stability of AAV circular intermediates suggests a novel linkbetween adenoviral helper functions and latent infection of AAV.

Aspects of inverted head-to-tail ITRs, which include palindromichairpins similar in structure to “Holliday-like” junctions, might impartrecombinagenic activity which aids in viral integration. Such Hollidayjunctions have been shown to play critical roles in directing homologousrecombination in bacteria through the processing of recombinationintermediates by RuvABC proteins (West, 1997; Lee et al., 1998).Interestingly, a mammalian endonuclease, analogous to bacterial RuvCresolvase, has also been isolated from cell lines (Hyde et al., 1994).Despite the theoretical considerations which might suggest that circularAAV genomes have characteristics of preintegration intermediates, astudy with recombinant retrovirus has demonstrated that palindromicLTR-LTR junctions of MMLV are not efficient substrates for proviralintegration (Lobel et al., 1989). Nonetheless, circular AAV genomes havebeen previously proposed as integration intermediates based on proviralstructure (Linden et al., 1996).

Example 2 Methods

Production of rAAV Shuttle Vector.

The cis-acting plasmid (pCisAV.GFP3ori) used for rAAV production wasgenerated by subcloning the Bsp1201/Not I fragment (743 bp) of the GFPtransgene from pEGFP-1 (Clontech) between the CMV enhancer/promoter andSV40polyA by blunt-end ligation. A 2.5 kb cassette containingbeta-lactamase and bacterial replication origin from pUC19 was bluntligated down-stream of GFP reporter cassette. The ITR elements werederived from pSub201.2 The entire plasmid contains a 4.7 kb AAVcomponent flanked by a 2 kb stuffer sequence. The integrity of ITRsequences was confirmed by restriction analysis with SmaI and PvuII, andby direct sequencing using a modified di-deoxy procedure which allowedfor complete sequence through both 5′ and 3′ ITRs. Recombinant AAVstocks were generated by co-transfection of pCisAV.GFP3ori and pRep/Captogether with co-infection of recombinant Ad.CMVlacZ in 293 cells. TherAV.GFP3ori virus was subsequently purified through 3 rounds of CsClbanding as described in Duan et al., 1997. The typical yields from thisviral preparation were 1012 DNA molecules/ml.

DNA titers were determined by viral DNA slot blot hybridization againstGFP ³²P-labeled probe with copy number plasmid standards. The absence ofhelper adenovirus was confirmed by histochemical staining of rAAVinfected 293 cells for beta-galactosidase, and no recombinant adenoviruswas found in 10¹⁰ particles of purified rAAV stocks. The absence ofsignificant wtAAV contamination was confirmed by immunocytochemicalstaining of rAAV/Ad co-infected 293 cells with anti-Rep antibodies.Transfection with pRep/Cap was used to confirm the specificity ofimmunocytochemical staining. No immunoreactive Rep staining was observedin 293 cells infected with 10¹⁰ rAAV particles.

Isolation of AAV Circular Intermediates from Muscle.

The tibialis anterior muscle of 4-5 week old C57BL/6 mice were infectedwith AV.GFP3ori (3×10¹⁰ particles) in Hepes buffered saline (30 μl). GFPexpression was analyzed by direct immunofluorescence of freshly excisedtissues and/or in formalin-fixed cryopreserved tissue sections in fourindependently injected muscles harvested at 0, 5, 10, 16, 22 and 80 dayspost-infection. Tissue sections were counter-stained with propidiumiodide to identify nuclear DNA. Hirt DNA (Hirt, 1967) (20 ml per musclesample) was isolated from at least three independent muscle specimen foreach time point and used to transform E. coli SURE cells using 3 ml ofHirt with 40 ml of electrocompetent bacterial (approximately 1×10⁹cfu/ug DNA, Strategene Inc.). The resultant total number of bacterialcolonies was quantified for each time point and the abundance ofhead-to-tail circular intermediates was evaluated for each time point(>20 bacterial clones analyzed) by PstI, AseI, SphI, and PstI/AseIdigestion, and confirmed by Southern blot analysis using ITR, GFP andstuffer probes. The head-to-tail configuration in typical clones werealso confirmed by dideoxy sequencing using primers EL118(5′-CGGGGGTCGTTGGGCGGTCA-3′; SEQ ID NO:1) and EL230(5′-GGGCGGAGCCTATGGAAAA-3′; SEQ ID NO:2) which are nested to 5′ and 3′ITR sequences, respectively. Zero hour controls were generated by mixing3×10¹⁰ particles of AV.GFP3ori with control uninfected muscle lysatesprior to Hirt DNA preparation. As described in Table 1, a number ofadditional controls for were performed to rule out non-specificrecombination of linear AAV genomes in bacteria as a source for isolatedcircular intermediates.

TABLE 1 Control Experiments for Rescue of Circular Intermediates inBacteria Number of Presence of Number Amp Head-to-Tail of ResistantCircular Type of Input Mole- Bacterial Intermedi- DNA Source of DNAcules Colonies ates^(e) Purified rAVV Hirt from Infected 3 × 10¹⁰approxi- Yes Muscle (22 day) mately  5 × 10³ Purified rAAV Virus 3 ×10¹⁰ 0 No reconstituted into Uninfected Muscle Hirt^(a) Linear ssDNAIsolated from 3 × 10¹⁰ 2 No Encompassing Purified Virus rAAV Genome^(b)Linear dsDNA Isolated from 3 × 10¹⁰ 3 No Encompassing proviral plasmidEntire rAAV (HindIII/PvuII)^(c) Genome Linear dsDNA Isolated from 3 ×10¹⁰ >6 × 10³ Yes Encompassing proviral plasmid Entire rAAV(HindIII/PvuII) Genome + ligase^(d) ^(a)Purified virus was reconstitutedinto muscle homogenates prior to preparation of Hirt DNA. ^(b)Viral DNApredominantly contained single stranded genomes as evident by Southernblot analysis against with ITR probe. However, small amount of dsDNA AAVgenomes also existed and are likely due to reannealing of singlestranded genomes during preparation. Purified viral DNA concentrationswere determined by OD₂₆₀ and 75 ng representing approximately 3 × 10¹⁰viral genomes were used for transformation of bacteria.^(c)HindIII/PvuII digestion was used to remove the entire rAAV genomefrom pcisAV.GFP3ori. HindIII and PvuII leave 10 and 0 bps of flankingsequence outside the 5′ and 3′ ITRs, respectively. The linear dsDNAfragment (4.7 kb) was gel isolated following blunting with T4 DNApolymerase and the DNA concentration determined by OD₂₆₀. One hundredand fifty ng of linear fragment representing approximately 3 × 10¹⁰viral genomes were used for transformation of bacteria. ^(d)Linear dsDNAviral genomes (HindIII/PvuII blunted fragment) were treated with T4 DNAligase prior to transformation of bacteria. ^(e)The presence ofhead-to-tail circular AAV intermediates were confirmed by restrictionenzyme digestion (AseI, PstI, and SphI) and Southern blotting againstITR probe.

Fractionation of Muscle Hirt DNA Preparations.

Preparative-scale fractionation of the muscle Hirt DNA was performed by1% agarose gel electrophoresis using the Bio-Rad Mini Prep Cell (Catalog#170-2908). A 4.5 ml (10.5 cm) tubular gel containing 1×TBE buffer waspoured according to manufacturer's specification. A total of 20 ml Hirtpreparation from one entire muscle sample was loaded on top of the gel.Electrophoresis was carried out at a constant current of 10 mA over aperiod of 5 hours. Sample eluent was drawn from the preparative gelapparatus by a peristaltic pump at a rate of 100 ml/min and eluted intoa fraction collector at 250 ml/fraction. The collected DNA wassubsequently concentrated by standard ethanol precipitation and used totransform SURE bacterial cells by electroporation as described above.

In Vitro Persistence of AAV Circular Intermediates.

Transgene expression and persistence of AAV circular intermediateplasmid clones were evaluated following transient transfection in Helaand 293 cells. Subconfluent monolayers of Hela cells in 24-well disheswere transfected with 0.5 mg of either AAV circular intermediates (p81or p87) or pCMVGFP using Lipofectamine (Gibco BRL Inc.). The cultureswere then incubated for 5 hours in serum free DMEM followed byincubation in DMEM supplemented with 10% fetal bovine serum. All plasmidDNA samples used for transfections were spiked with pRSVlacZ (0.5 mg) asan internal control for transfection efficiency. At 48 hourspost-transfection, cells were passaged at a 1:10 dilution and allowed togrow to confluency (day 5), at which time GFP clones were quantified forsize and abundance using direct fluorescent microscopy. The percent ofbeta-galactosidase-expressing cells was also quantified at this timepoint by X-gal staining. At 5 days, cells were passaged an additionaltime (1:15 dilution) GFP clones were quantified again at day 10. Thepersistence of plasmid DNA at passage-5, 7, and 10 dayspost-transfection was evaluated by Southern blot analysis of totalcellular DNA using ³²P-labeled GFP probes. To determine whether thehead-to-tail ITR array within circular intermediates was responsible forincreases in the persistence of GFP expression, the head-to-tail ITR DNAelement was subcloned into the pGL3 luciferase plasmid to generatepGL3(ITR). The head-to-tail ITR DNA element was isolated from a monomercircular intermediate (p81) by AatII and HaeII double digestion andsubsequently inserted into the SalI site of pGL3 (Promega) by bluntligation. The resultant plasmid pGL3(ITR) contains the luciferasereporter and head-to-tail ITR element 3′ to the polyA site. Theintegrity of the ITR DNA element within this plasmid was confirmed bysequencing. The persistence of transgene expression from pGL3(ITR) wascompared to that of pGL3 by luciferase assays on transiently transfectedHela cells as described above and analyzed at 10 days (passage-2).Transfection efficiencies were normalized using a dual renillaluciferase reporter vector (pRLSV40, Promega).

Results

AAV Circular Intermediates Represent Stable Episomal Forms of Viral DNAAssociated with Long-Term Persistence of Transgene Expression in Muscle.

To evaluate the molecular characteristics of rAAV genomes in muscle, arAAV shuttle viral vector (AV.GFP3ori) was utilized which harbors anampicillin resistance gene, bacterial origin of replication, and GFPreporter gene (FIG. 1A). This recombinant virus was used to evaluate thepresence of circular intermediates by bacterial rescue of replicationcompetent plasmids. In these studies, delivery of AV.GFP3ori (3×1010particles) to the tibialis muscle of mice led to GFP transgeneexpression which peaked at 22 days and remained stable for at least 80days (FIG. 4A). These results confirmed previous successes in rAAVmediated gene transfer to muscle (Kessler et al., 1996; Herzog et al.,1997; Xiao et al., 1996; Clark et al., 1997; Fisher et al., 1997). Theformation of circular intermediates was evaluated by E. colitransformation of Hirt DNA harvested from muscle at 0, 5, 10, 16, 22,and 80 days post-infection with AV.GFP3ori.

In these muscle samples, circular intermediates were found to have acharacteristic head-to-tail structure with 1-2 ITR repeats. The mostabundant form included two inverted ITRs within a circularized genome(FIG. 4B, clone p17). This figure also depicts a less frequent form(<5%) of circular intermediates observed, p439, with undeterminedstructure. When this type of replication competent plasmid was seen, itwas not included in the quantification of head-to-tail circularintermediates since its structure could not be conclusively determined.The total abundance of muscle Hirt derived head-to-tail circularintermediates (with 1-2 ITRs) demonstrated a time-dependent increasethat peaked with transgene expression at 22 days and slightly decreasedby day 80 (FIG. 5A). Increased diversity in the length of ITR arrayswithin circular intermediates was seen at longer time points. Forexample, FIG. 5B demonstrates several isolated circular intermediateswith 1-3 ITRs isolated from 80 days muscle Hirt samples. This is incontrast to the more uniform structure of circular intermediates withtwo ITRs in a head-to-tail conformation at 5-22 days post-infection.

To evaluate the potential for artifactual rescue of linear rAAV genomesby recombination in bacteria, several control experiments wereperformed. First, uninfected control muscle Hirt preparations, spikedwith an equal amount of rAAV virus used for in vivo infection ofmuscles, failed to give rise to replicating plasmids followingtransformation of E. coli. Second, when a blunted linear double strandedHindIII/PvuII fragment isolated from pcisAV.GFP3ori (encompassing theentire rAAV genome) was used to transform bacteria, no ampicillinresistant bacterial colonies were obtained. The addition of T4 ligase tothis fragment, however, led to significant numbers of bacterialcolonies. Third, when purified single stranded rAAV DNA was used fortransformation, no bacterial colonies were obtained. As summarized inTable 1, these results confirm that in the absence of productiveinfection, rAAV genomes themselves are incapable of recombining intoreplication competent plasmids in bacteria. Hence, in vivocircularization of rAAV genomes is a prerequisite for rescuingautonomously replicating plasmids in E. coli with this shuttle vector.

Molecular Weight of Circular Intermediates Suggest a Conversion fromMonomer to Multimer Forms Over Time.

To further characterize the circular intermediates isolated frommuscles, Hirt samples from 22 days and 80 days post-infected muscleswere size fractionated by continuous-flow gel electrophoresis (BioRad).As shown in FIG. 6, the majority of circular intermediates at 22 dayspost-infection size fractionated at a molecular weight of less than 3Kbp. Very few clones were isolated from fractions between 3 to 5 kb andno clones were obtained from fractions larger than 5 kb at this timepoint. Furthermore, this size fractionated molecular weight of in vivoHirt derived circular intermediates at 22 day time points correlatedwith that of head-to-tail monomer undigested circular intermediateplasmids rescued in bacteria from this same time point (approximately2.5 kb). These data suggest that at early time points post-infection inmuscle, the predominant form of circular intermediates likely occurs asmonomer genomes. The lower mobility of this fraction as compared toreplication form monomer (Rfm=4.7 kb) and dimer (Rfd=9.4 kb) genomesprovides indirect evidence that these forms are not responsible forrescued plasmids in these Hirt samples. Interestingly, when 80 daymuscle Hirt samples were size fractionated, more clones were retrievedfrom higher molecular weight fractions ranging from 3-12 kb (FIG. 6).This shift in the molecular weight of circular intermediates indicatesthe potential for recombination between monomer forms in the generationof large circular multimer genomes. Such concatamerization has beenpreviously observed in muscle and has traditionally been hypothesized toinvolve linear integrated forms of the AAV genome (Herzog et al., 1997;Xiao et al., 1996; Clark et al., 1997; Fisher et al., 1997). This datasheds new light on the molecular characteristics of these persistent AAVgenomes and suggests that they are in fact circular and episomal. Basedon yields of retrievable circular plasmids reconstituted in Hirt DNA,the efficiency of bacterial transformation, and the initial innoculum ofvirus, we estimate that approximately 1 in 400 viral DNA particlescircularize following infection in muscle (Table 2).

TABLE 2 Yield of Circular Intermediate Isolation from Hirt DNA StartingNumber Actual Bacterial of Plasmid or Number Adjusted Transformation AAVGenomes of Amp^(r) cfu Yield Hirt DNA from rAAV 3 × 10¹⁰ molecules 5 ×10³ cfu 5 × 10⁵ cfu^(e) Infected Muscle ^(a) Hirt DNA + 230 ng 3 × 10¹⁰molecules 2 × 10⁶ cfu^(d) 2 × 10⁸ cfu LacZ Plasmid ^(b, c) 230 ng LacZPlasmid ^(c) 3 × 10¹⁰ molecules 2 × 10⁸ cfu — ^(a) The actual amount ofHirt used for transformation was 3/20 the entire Hirt DNA. The numbershave been adjusted to reflect viral innoculum and yields for the entiremuscle. ^(b) Plasmid DNA was spiked into mock infected musclehomogenates prior to isolation of Hirt DNA. This reconstituted Hirt DNAwas then used for transformation of bacteria. ^(c) The actual microgramamounts of plasmid used in reconstitution experiments was 10 ng. Thenumbers have been adjusted for comparison to normalize the number ofplasmids genomes to that used in AAV experiments. Control LacZ plasmidwas approximately 7000 bp with a molecular weight of 4.6 × 10⁶ g/mole.^(d) The average of several experiments indicates an approximate100-fold reduction in the number of cfu recovered from bacterialtransformations with DNA isolated from Hirt extract spiked with plasmidsas compared to transformation with an equivalent amount of plasmid DNAalone. ^(e) Adjusted yield indicate approximately 1 in 400 AAV genomescircularize in vivo. Given the fact that not all rAAV particles likelycontain functional DNA molecules and intermediates may integrate, thesecalculations may represent an underestimation.

AAV Circular Intermediates Demonstrate Increased Persistence as PlasmidBased Vectors.

Based on the finding that circular AAV intermediates were associatedwith long term persistence of transgene expression in muscle, rAAVcircular head-to-tail intermediates may be molecular structures of theAAV genome associated with the latent life cycle and increased episomalstability. Several aspects of the structure of AAV circularintermediates may account for their increased stability in vivo. First,circularization of AAV genomes may create a nuclease resistantconformation. Secondly, since the only viral sequences contained withincircular intermediates are the head-to-tail ITR array, these sequencesmight bind cellular factors capable of stabilizing these structures invivo. Several studies have demonstrated increased persistence oftransgene expression with plasmid DNA encoding viral ITRs (Philip etal., 1994; Vieweg et al., 1995). The results described above provide afunctional explanation for the increased persistence through theassociation with circular intermediate formation as part of the AAV lifecycle.

To more closely evaluate the persistence of AAV head-to-tail circularintermediates, several in vitro experiments were performed bytransfecting these intermediates into Hela cells and assessing thestability of plasmid DNA and transgene expression by GFP clonalexpansion. Results from Hela cell transfection experiments demonstratedthat two monomer head-to-tail circular intermediates (p81 and p87)studied gave rise to a 10-fold higher number of five and ten daytransgene-expressing clones, as compared to a control pCMVGFP plasmidlacking the ITR sequences (FIGS. 7A and B). Additionally, the size ofGFP positive colonies at 5 days post-transfection was three-fold largerin Hela cells transfected with p81 and p87, as compared to the pCMVGFPcontrol vector (FIGS. 7A and B). These studies suggest the AAV circularintermediates have increased stability of transgene expression andsubstantiate findings in muscle.

To confirm the increased molecular persistence of head-to-tail circularintermediates following transfection into Hela cells, total DNA (low andhigh molecular weight) was isolated from cultures of pCMVGFP and p81transfected Hela cells at various passages post-transfection andanalyzed by Southern blotting. Southern blots hybridized to ³²P-labeledGFP probes demonstrated a significantly higher level of p81 plasmid DNAat passage-7 as compared to the control vector lacking the head-to-tailITR sequence (FIG. 7C). The majority of signal in undigested DNA sampleswas associated with a 4.7 kb band migrating at the approximate size ofthe uncut monomer plasmids. Together with the fact that the majority ofsignal from all cell cultures in FIG. 7C disappeared by passage-10,these data suggest that these plasmids predominantly remained episomal.Thus, in both muscle and Hela cells, increased persistence of AAVcircular intermediates is correlated with stable transgene expression.

ITR Arrays are Responsible for Increased Persistence.

To investigate whether the head-to-tail ITR DNA element was responsiblefor the increased persistence of circular intermediates, we cloned thisDNA element into a secondary luciferase vector (pGL3) to give rise topGL3(ITR). Transient transfection experiments in Hela cells demonstrateda five-fold increase in the persistence of luciferase expression inserially-passaged cultures at 10 days in pGL3(ITR) as compared to thatof pGL3 transfected (FIG. 7D). These findings support the hypothesisthat the head-to-tail ITR DNA element contained within circularintermediates is responsible for mediating the increased persistence oftransgene expression and suggest a mechanism by which these molecularintermediates may confer stability to AAV genomes in vivo. Furthermore,increases in the stability of transgene expression conferred by thiselement appear to be primarily context independent, since thehead-to-tail ITR element was 3′ to the luciferase gene in pGL3(ITR) and5′ to the GFP transgene in AAV circular intermediates.

Discussion

Characterization of integrated proviral structures in different celllines has demonstrated head-to-tail genomes as the predominantstructural forms for both wild type and recombinant AAV (McLaughlin etal., 1988; Cheung et al., 1980; Duan et al., 1997). This is in contrastto the head-to-head and tail-to-tail structures observed in AAVreplication intermediates (Rfm and Rfd). Both Rfm and Rfd configurationshave also been demonstrated in rAAV infected cells and enhancedconversion of ssAAV genomes to double stranded Rfm and Rfd forms hasbeen suggested as a mechanism for augmentation of rAAV transduction byadenovirus in cell lines (Ferrari et al., 1996; Fisher et al., 1996).However, it is plausible that the mechanisms responsible for theformation of Rfm and Rfd molecules are different from pathways whichlead to long-term transgene expression. In support of this hypothesis isa recent study evaluating augmentation of rAAV transgene expression byadenovirus in liver (Snyder et al., 1997). These studies havedemonstrated that co-infection of the liver with adenovirus and rAAVenhances short term transgene expression while long term expression wasno different than rAAV alone. The exact mechanism for the formation ofhead-to-tail circular intermediates is not clear, however similarstructures have been demonstrated to act as pre-integrationintermediates for retrovirus (Varmus, 1982). In this regard,circularized retroviral genomes with one and two viral LTRs have beenproposed. In addition, circular pre-integration intermediates have alsobeen suggested by recent studies on wtAAV integration (Linden et al.,1996b). The demonstration that circular intermediates exist in rAAVinfected muscle explains several features of latent phase infection withrAAV vectors including proviral structure and stable episomalpersistence.

Previous studies have suggested that rAAV genomes delivered to musclemight persist as head-to-tail concatamers (Herzog et al., 1997; Clark etal., 1997; Fisher et al., 1997). However, it is currently unknownwhether these concatamers exist as free episomes or as integratedproviruses in the host genome. The results described above, i.e.,demonstrating prolonged persistence of head-to-tail circularintermediates at 80 days post-infection, suggest that a large percentageof rAAV genomes may remain episomal. The conversion of monomercircularized genomes to larger circularized multimers appears to be anaspect associated with long term persistence and likely representsrecombinational events between monomer intermediates. Although thebacterial rescue strategy was not capable of satisfactorily addressingthe size of multimers, our modified approach to size fractionating HirtDNA prior to bacterial rescue of intermediates lends support to thishypothesis. Additional supportive evidence for increased recombinationover time is the finding that greater variability in the length of ITRarrays was observed at longer time points post-infection. For example,at 5-22 days the majority of circular intermediates contained 2 ITRs ina head-to-tail fashion. This is in contrast to 80 day time points wherethe lengths of ITR arrays ranges from 1-3 ITRs. Such diversity of ITRarrays in muscle infected with AAV has been previously found using PCRapproaches (Herzog et al., 1997; Fisher et al., 1997). In addition, the30% decline in the abundance of circular intermediates in muscle between22 and 80 days also supports a hypothesis that these molecular forms ofAAV may represent pre-integration complexes.

Given the fact that circular intermediates had long term persistence inmuscle, certain structural features of these intermediates may affectepisomal stability of DNA. Previous studies have noted increasedpersistence of transgene expression from plasmids encoding AAV ITRs(Philip et al., 1994; Vieweg et al., 1995). However, the physiologicsignificance of this finding has remained elusive. The present study,demonstrating the head-to-tail ITR arrays isolated from AAV circularintermediates can confer increased episomal persistence to plasmidsfollowing transfection in cell lines, gives a mechanistic framework forITR effects on plasmid persistence. Furthermore, the correlation thatAAV circular intermediates have increased persistence in cell lines invitro, lends support to the hypothesis that these structures representstable episomal forms following rAAV transduction in muscle. Stabilityof circular intermediates in vivo might be mediated by the binding ofcellular factors to “Holliday-like” junctions in ITR arrays whichstabilize or protect DNA from degradation.

rAAV has been shown to be an efficient vector for expressing transgenesin various tissues in addition to muscle, such as brain, retina, liver,lung, and hematopoetic cells (Snyder et al., 1997; Muzyczka, 1992;Kaplitt et al., 1994; Walsh et al., 1994; Halbert et al., 1997; Koeberlet al., 1997; Conrad et al., 1996; Bennett et al., 1997; Flannery etal., 1997). Despite these advances in the application of rAAV, themechanisms of in vivo rAAV-mediated transduction and persistence oftransgene expression still remain unclear. Such questions as to themolecular state of rAAV following in vivo delivery is highly relevant tothe clinical application of this viral vector. For example, should rAAVprimarily persist as an randomly integrated provirus, the potential forinsertional mutagenesis could present a major theoretical obstacle inthe use of this vector due to the potential for mutational oncogenesis.The demonstration that rAAV can persist as episomes suggest that randomintegration and associated risks of malignancy may not be a majorconcern for this viral vector system. Additionally, the moleculardeterminants of AAV circular intermediates associated with increasedpersistence in cell lines appear to be contained within the DNA elementsencompassing the inverted ITRs. The isolation of this naturallyoccurring viral DNA element, which forms as part of the AAV life cycleand acts to stabilize circular episomal DNA, may prove useful inincreasing the efficacy of both viral and non-viral gene therapyvectors.

Example 3 Evidence for Increased Episomal Persistence of AAV CircularIntermediates in a Model for In Utero Plasmid-Based Gene Therapy

Persistence of AAV circular intermediates were assessed by injection ofplasmid DNA directly into the pronucleus of fertilized Xenopus oocytes.Twenty-five ng of the p81 isolate of AAV circular intermediates wasinjected at the single cell stage of fertilized Xenopus oocytes. Thisplasmid was compared to the proviral plasmid pCisAV.GFP3ori, whichcontains two ITRs separated by stuffer sequence in an alternativeconfirmation to ITRs in p81. FIG. 13 depicts the persistence of GFPplasmids as assessed by direct fluorescence of GFP. At this state oftadpole development, the fertilized oocyte has expanded from a singlecell to approximately 10⁶ cells.

These studies confirm that AAV circular intermediates (p81) confer ahigher level of stability in development Xenopus oocytes than plasmidscontaining similar transcriptional elements and ITR sequences in analternative confirmation. Given that in the case of p81 injectedoocytes, tadpoles are completely fluorescent, the data suggests thatsome level of integration may have occurred.

Example 4 Liposome Mediated Transfer of Vectors of the Invention to theAirway and Muscle

Studies evaluating the mechanisms of recombinant adeno-associated virus(AAV) transduction have identified a novel molecular intermediateresponsible for episomal persistence. This intermediate is characterizedby a circularized AAV genome with head-to-tail ITR repeats. Circularintermediates of rAAV were identified using a recombinant shuttle vectorcapable of propagating circularized viral genomes in bacteria. Pivotalexperiments in cell lines demonstrate that the formation and persistenceof these circular intermediates are augmented in the presence of helperadenovirus. These findings suggest that cellular factors induced byadenoviral gene expression may modulate both the formation and/orpersistence of AAV circular intermediates. Furthermore, studies inmuscle have demonstrated that following rAAV infection, the formationand persistence of AAV circular intermediates correlates with the onsetand maintenance (at 80 days) of transgene expression, respectively.Moreover, a 300 bp fragment encompassing the head-to-tail inverted ITRrepeats found in AAV circular intermediates when cloned intoheterologous expression plasmids can confer increased stability to thoseplasmids in HeLa cells. The structural aspects of AAV circularintermediates may lead to development of non-viral, plasmid based, genetransfer vectors with increased persistence of transgene expression.

To determine whether AAV circular intermediates which differ in lengthand/or sequence of the ITR array are more efficacious plasmid basedvectors for liposome-mediated gene transfer to the airway and muscle,several distinct forms of AAV circular intermediates are evaluated asplasmid-based delivery systems in three model systems of the airwayincluding: 1) in vitro polarized primary airway epithelial monolayers,2) mouse lung, and 3) human bronchial xenografts. Persistence isevaluated at both the level of transgene expression (using GFP andluciferase reporters) and at the level of episomal and integratedtransgene derived DNA. Studies are performed to assess whetherintegration can be specifically enhanced by co-transfection with Rep DNAor mRNA. These studies also evaluate both the extent of integration andsite specificity to AVS1 sites in chromosome 19 of human model systems.

Gene therapy using plasmid-based delivery systems have encounteredseveral obstacles to efficient transgene expression. These obstaclesinclude transient expression of transgenes and rapid degradation of DNA.In contrast, viruses have developed efficient mechanisms for transducingcells and expressing encoded viral genes. The molecular characteristicsof AAV circular intermediates which confer increased persistence oftransgene expression include a DNA element encompassing the head-to-tailITR. Based on the findings that circular intermediates have increasedepisomal persistence in muscle following rAAV transduction, thesestructures may also have increased persistence as plasmid-based vehiclesto the airway. Interestingly, several naturally occurring mutationswhich are found in approximately 50% of AAV circular intermediatesaffect the stability of the intermediate.

Several findings evaluating the efficiency of AAV circular intermediateformation from recombinant viral vectors have suggested that thesestructures are augmented in abundance by the presence of the E2aadenoviral gene product. These molecular structures may representpreintegration intermediates which, in the case of wild-type AAV, wouldefficiently integrate into the cellular genome by Rep facilitatedmechanisms. However, in the case of recombinant AAV genomes (in theabsence of Rep proteins), evidence suggests that these structures haveincreased episomal stability. To test whether exogenous addition of Repand/or E2a can increase the efficacy of AAV circular intermediates bymodulating their stability and/or integration, co-transfection methodswith Rep encoding plasmids and mRNA are conducted. Additionally,exogenously supplied E2a DNA binding protein (DBP) may also enhancestability of AAV circular intermediates. Rep may increase theintegration of circular intermediates while E2a may increase theirepisomal stability. Several observations including the association ofE2a DBP with AAV genomes in the nucleus support a direct interactionbetween DBP and AAV circular intermediates. Furthermore, if DBPassociates with AAV circular intermediates, its encoded nuclearlocalization sequence (NLS) may enhance nuclear sequestration of theseplasmids in the nucleus. Alternatively, E2a may act to alter thepersistence of AAV circular intermediates through the induction ofcellular factors which interact with the ITR array.

Liposome mediated gene transfer to the airway has considerableadvantages due to the low level of toxicity. However, limitationsinclude transient low level expression in differentiated airwayepithelia. Despite this apparent limitation, several laboratories havehad considerable success with the use of cationic liposome-mediated genetransfer in several animal models including mouse and rat lung, andnumerous laboratories have pursued clinical trials, which suggested thatthese vehicles may show promise for gene therapy of the cystic fibrosis(CF) lung. Thus, delivery of the present vectors in plasmid form vialiposomes may be a safe and effective vehicle for gene transfer to theairway.

To assess whether AAV circular intermediates may also have increasedpersistence in airway epithelial cells as seen in Hela cells, severaldistinct forms of circular intermediates delivered by liposome-mediatedtransfection into primary airway epithelial cells, are evaluated. Basedon the diversity of ITR repeat elements between various isolatedcircular intermediates (i.e., including 0, 1, 2, and 3 ITRs), circularintermediates isolated from later time points in muscle may have beennaturally selected for increased stability in vivo. Hence, thestructural consistencies between AAV circular intermediates areidentified which give increased persistence as plasmid based vectors forgene transfer.

Circular intermediates containing the GFP reporter gene and 1, 2, and 3ITRs are transfected into primary airway cultures and polarizedepithelial cell monolayers using the cationic lipid GL-67 (GenzymeInc.). DNA to lipid ratios are optimized using a luciferase reporter.Additionally, the addition of EGTA, or the use of calcium-free media,can increase the extent of gene transfer about 10-fold, and may beincluded to enhance gene transfer to polarized epithelial monolayers. Toevaluate persistence and expression of transgenes from circularintermediates, direct fluorescent microscopy and Southern blotting ofboth Hirt and genomic DNA with GFP P³²-labeled probes are utilized.Proliferating cultures of primary airway epithelial cells can bepassaged up to 4 times during this analysis. In contrast, polarizedepithelial monolayers are evaluated at 1 week intervals for DNApersistence for up to 6 weeks. Since GFP transgene expression may be lowand difficult to detect by direct fluorescence, GFP is quantitated byfluorometer of cell lysates.

Following AAV transduction, circular intermediates may form within cellsand certain structures of these intermediates may persist by virtue ofaffinity for cellular factors which bind at ITR arrays. If this is true,then it may be possible to select for and isolate optimal circularintermediates with increased persistence in airway cells by batchscreening of circular intermediates pools from rAAV infected airwayepithelia.

Primary airway epithelia cell cultures are infected with AV. GFP3ori(MOIs of 1000 to 10,000 DNA part/cell) and low molecular Hirt DNA isprepared at 5-15 days post-infection. Hirt DNA containing circularintermediates from rAAV infected cells is used to then transfect primaryairway epithelial cells from which Hirt DNA is prepared at 5-15 dayspost-transfection. This second Hirt isolation is then used to isolatereplication competent plasmids following transformation into bacteria.This selection process may give rise to those populations of circularintermediates with increased episomal persistence in airway epithelialcells. Selected clones of circular intermediate plasmids isolated bythis procedure are then tested individually for increased persistencefollowing liposome mediated transfection. These studies are performed ina batch type screening in 24 well plates using two serial passages forpersistence. Once plasmids having increased persistence are isolated,their structure and sequence of ITR arrays are characterized. Sincescreening is performed on small-scale cultures, it may be necessary toimplement semi-quantitative screening for DNA persistence within thefirst round of transfection using PCR methods. Candidate plasmids with ahigh level of increased persistence as compared to control plasmidswhich lack ITR sequences but contain the identical promoter-reporterelement, are evaluated on a larger scale transfection amenable toanalysis by Southern blotting of total DNA.

To evaluate selected circular intermediate structures in vivo, twomodels including mouse lung and the human bronchial xenograft areemployed. 10 wk BalbC mice are transfected with GL-67/DNA complexes at aratio of 25 μg plasmid/25 μg lipid in an iso-osmotic solution ofDextrose. At 1, 5, 10, 15, and 20 days post-transfection lungs of miceare harvested for immunofluorescent detection of GFP in formalin fixedsections and for quantitative fluorometry of tissue lysates. Southernblots are employed to evaluate the persistence of plasmids in Hirt andgenomic DNA. In addition to evaluating the persistence of selectedcircular intermediates which have the highest level of persistence within vitro models, luciferase constructs are evaluated in which the ITRarray has been cloned either 5′ or 3′ to the reporter gene. Furthermore,the use of luciferase reporters allows for more sensitive assessment oftransgene activity in cell lysates.

Similarly, in vivo persistence of transfected circular intermediates andheterologous plasmids containing ITR arrays found within circularintermediates is evaluated in human bronchial xenografts.

Findings evaluating the effects of adenoviral co-infection on circularintermediate formation and persistence have suggested that E2a DBP leadsto a 10-fold increase in the abundance of circular intermediates ascompared to E2 deleted virus. Furthermore, studies with E1-deleted virushave demonstrated that the persistence of circular intermediates in Helacells is increased at 72 hours post-infection. These studies suggestthat E2a DBP may augment circular intermediate formation and/or increasethe stability of these structures by an unknown mechanism. E2a DBP mayinteract directly with circularized genomes and/or induce cellularfactors which interact with sequences in these AAV genomes. Since DBPencodes an NLS, this protein may act to shuttle circular intermediatesto regions of nucleus that allow for increased stability of thesestructures. NLS sequences have been shown to cooperatively interact withnucleolar targeting sequences and hence we will also evaluate ifsubnuclear targeting is important in maintaining the increased stabilityof circular intermediates containing ITR arrays. Furthermore, it iscurrently unknown where circular intermediates form in the cell and itremains plausible that they may form in the cytoplasm or nucleus. Hence,if DBP associates directly with circular intermediates, it may act as anNLS for DNA to enter the nucleus as well.

Several in vitro reconstitution models are used to investigate theinteraction of circular intermediates with DBP and their affect on invivo persistence following DNA transfection in Hela cells. Furthermore,results evaluating the affects of various mutant adenoviral vectors oncircular intermediate and Rfm/Rfd formation have suggested that thesetwo types of intermediates occur by independent pathways indicative oflatent and lytic infection, respectively. In the setting of wild typeAAV, circular intermediates may be pre-integration complexes, which inthe presence of Rep, efficiently integrate into the host genome. Incontrast, in the absence of Rep, circular intermediates may accumulateepisomally in rAAV infected cells. To this end, methods of supplementingRep function may be capable of enhancing integration of plasmid baseddelivery of AAV circular intermediates. Thus, experiments in whichco-transfection of circular intermediate plasmids with Rep expressionplasmids or mRNA are conducted.

To investigate whether DBP can augment the stability of circularintermediates by increasing targeting to the nucleus, a Hela cell line(gmDBP6) is utilized which encodes an inducible E2a gene under adexamethasone responsible element. This cell line gives rise to highlevels of DBP in nuclear extracts by Western blot following treatmentwith dexamethasone. gmDBP6 cells (+/−DEX) are transfected with variousAAV circular intermediate plasmids containing 0, 1, 2, and 3 ITRs andtotal cellular and nuclear plasmid content evaluated by subcellularfractionation using Southern blotting against GFP probes. The timecourse of these studies is initially within the range of 12 hours to 4days post-transfection. Transgene expression is evaluated by fluorometry(in cell lysates), and fluorescent microscopy (in viable cells), for GFPand luminescence for luciferase. Hela cells have demonstrated thatimmediate increases in transgene expression from AAV GFP circularintermediates as compared to control GFP plasmids occur as early as 24hours post-transfection. Thus, certain cellular factors may facilitatean immediate accumulation of circular intermediates in the nucleus. DBPmay invoke this increase by either direct interactions with ITRsequences or by the induction of cellular factors. To evaluate thepotential for direct interactions between DBP and circularintermediates, various form of ITR arrays found within circularintermediates are end-labeled with γ-ATP³² and evaluated for binding byelectrophoretic mobility shift assays to nuclear extracts from gmDBP6cells (+/−DEX). Supershifts, with DBP antibodies and competitionexperiments with cold ITR sequences and non-specific DNA, are used ascontrols for specific binding.

In a second model system aimed at evaluating the potential of DBP forshuttling and/or sequestering of circular intermediates to the nucleus,microinjection experiments in oocytes are performed with 50 ng ofplasmid DNA of circular intermediates with and without 50 ng of DBPmRNA. Experiments initially evaluate the time course of GFP transgeneexpression (+/−DBP cRNA) by direct fluorescent microscopy. If majordifferences are seen, quantitative fluorometry of individual wholeoocytes in 96 well plates is conducted. Similar studies on nucleartargeting in the presence of DBP can also be evaluated in this model bypooling microinjected oocytes for nuclear isolation and Southern blotanalysis.

A third experimental model to evaluate nuclear targeting and/oraccumulation of circular intermediate vectors in the presence andabsence of DBP involves the microinjection of fluorescently labeledplasmid DNA into the cytoplasm and real time imaging to follow thenuclear accumulation of DNA. The DNA fluorescent dye, TOTO-1, is used tolabel DNA prior to injection. This dye forms an extremely stable complexwith negligible diffusion and re-incorporation into nuclear DNAfollowing transfection into polarized airway epithelial cell monolayers.Co-localization of DBP with wtAAV DNA genomes at focal hot spots withinthe nucleus supports the observation that nucleolar targeting may beimportant for persistence. These experiments are also performed inprimary airway epithelial cells and in vivo models of the airway byeither co-transfection of circular intermediates with DBP expressingplasmids and/or mRNA.

The effects of Rep co-transfection on the integration of circularintermediate plasmids is also evaluated. Two methods are used to expressRep including: 1) co-transfection with Rep expressing plasmids, and 2)co-transfection with Rep encoding mRNA. Initially, Hela, CFT1, and IB-3cells are tested, as transformed cells may be more amenable to expansionand evaluation of integration. Both CFT1 and IB-3 cells represent airwayepithelial cells. Experiments are performed by cationic liposome (GL-67)mediated transfection of circular intermediate DNA with varying doses ofa Rep-containing expression vector, e.g., pCMVRep. The extent ofintegration is also evaluated by two criteria, Southern blotting of Hirtand genomic DNA and clonal expansion of GFP expressing cells. SinceSouthern blot has an approximate limit of sensitivity of 1 integratedplasmid molecule per 10 cellular genomes, clonal expansion may benecessary to evaluate persistence in less transfectable cells such asCFT1 and IB-3 cells. Cell lines are evaluated over the course of 1-10passages.

Sustained expression of Rep by plasmid mediated co-transfection may betoxic to cells, hence co-transfection with Rep mRNA is also evaluated.Cationic liposome:mRNA mediated transfection has been previously shownto work in cell lines and although the level of expression is much moretransient than for DNA, in these studies it may be an advantage. Initialstudies are performed with in vitro transcribed Rep mRNA alone toevaluate the μg amount of mRNA needed for Rep expression as determinedby Western blot. Once the threshold for detectable Rep expression isestablished, increasing amounts of Rep mRNA are co-transfected withcircular intermediate DNA. Similar assays are used as described above toevaluate the extent of AAV circular intermediate integration. Iffindings suggest that increased integration if facilitated by Rep, thesite specificity of this integration can be evaluated by cloning GFPexpressing cells after the 10th passage by serial dilution. These GFPexpressing clones are expanded and genomic Southern blots assessed withboth GFP and AVS1 specific probes. By evaluating a number of restrictionenzymes which either do not cut or cut once within the circularintermediate plasmid, it will be determined whether integration hasoccurred at the AVS1 loci.

To test whether secondary structure rather than primary sequence is theimportant determinant of increased episomal stability of AAV circularintermediates, synthetic DNA sequences are generated with identicalsecondary structure to several ITR arrays in circular intermediates. Theprimary sequence is completely altered and bares no resemblance tosequences contained within native AAV ITRs. These synthetic DNAsequences are tested for their ability to confer increased episomalstability to heterologous plasmids in several model systemsincluding: 1) the airway, 2) muscle, 3) and developing Xenopus embryos.The developing Xenopus embryo model is ideal for testing integration andpersistence of plasmid based vectors for application of in utero genetherapy. If synthetic DNA sequences with similar secondary structure toITRs are found to confer increased persistence to plasmid based vectors,then determinants for protein binding which facilitate persistence areindependent of primary base sequence. These studies allow theoptimization of the secondary structural requirements by synthesizing awide range of DNA molecules with varying degrees of palindromic repeats.Furthermore, the secondary structure may not bind proteins directly butfacilitate recombination of plasmids to large concatamers which haveincreased episomal stability or enhanced integration efficiencies.

Example 5 Delivery of Multiple Genes through IntermolecularConcatamerization Methods Recombinant AAV Vectors.

Two rAAV vector stocks were generated for use in these studies,AV.GFP3ori (Example 1) and AV.Alkphos (also known as CWRAPSP, a gift ofDusty Miller) (Halbert et al., 1997). Virus stocks were generated byco-transfection of 293 cells with either pCisAV.GFP3ori or pCWRAPSPalong with pRep/Cap, followed by co-infection with recombinantAd.CMVlacZ helper virus (Example 2). rAAV was then purified throughthree rounds of CsCl density gradient centrifugation as previouslydescribed by Duan et al. (1997). Purified viral fractions were heated at60° C. for 1 hour to inactivate any residual contaminating helperadenovirus. The yields for AV.GFP3ori and AV.Alkphos were 1×10¹² and7×10¹¹ particles per ml, respectively, as determined by slot blothybridization with ³²P-labeled GFP or Alkphos probes. Infectious titersdetermined by infection of 293 cells with rAAVs were 1.1×10⁹ IU/ml(AV.GFP3ori) or 8.6×10⁸ IU/ml (AV.Alkphos). Controls testing forcontamination of rAAV stocks with wtAAV by anti-Rep immunocytochemicalstaining in rAAV/Ad.CMVlacZ co-infected 293 cells were negative (limitof sensitivity is less than 1 infectious wtAAV particle per 10¹⁰ DNAparticles of rAAV). Similarly, histochemical staining forβ-galactosidase in rAAV infected 293 cells showed no detectablecontamination with helper adenovirus in 10¹⁰ DNA particles of rAAV(limit of sensitivity).

Infection of Muscle Tissue and Evaluation of Transgene Expression.

The C57BL/6 mice used for these experiments were housed in a virus-freeanimal care facility and were maintained under strict University of Iowaand NIH guidelines, using a protocol approved by the Animal Care and UseCommittee and facility veterinarians. Four to five week old micereceived bilateral 30 μl injections of a mixture of both AV.GFP3ori andAV.Alkphos into the tibialis anterior muscle (5×10⁹ DNA particles ofeach virus per muscle). Controls included uninjected muscles and musclesreceiving injections of one of the viruses alone. At 14, 35, 80, and 120days post-infection, animals were euthanized and tissues were harvestedfor evaluation of transgene expression and preparation of low molecularweight Hirt DNA. For each experimental time point, at least 3independently injected muscles were evaluated.

In all experiments, GFP fluorescence was visualized in freshly excisedmuscle tissue prior to processing. A portion of the same muscle wasfixed with 2% paraformaldehyde in phosphate buffered saline, andcryoprotected in graded sucrose solutions before embedding in optimalcutting temperature medium (OCT). Sections (6 μm) were then evaluatedfor GFP expression directly and Alkphos expression following heatinactivation of endogenous Alkphos and histochemical staining forAlkphos activity (Engelhardt et al., 1995). To confirm dual localizationof GFP and Alkphos expression in the same muscle fibers, either serialsections were evaluated for GFP and Alkphos expression or the samesection was first photographed for GFP expression followed byhistochemical staining for Alkphos and re-imaging of the same field.

Rescue of Circular Intermediates from Muscle Hirt DNA.

Low molecular weight Hirt DNA was prepared from 20 mg specimens ofinjected muscles from 3 animals at each time point (Example 2). Hirt DNA(4 μl; ⅕ of the total volume) was then used to transform 50 μl ofelectrocompetent SURE cells (Stratagene) using a BioRad E. colielectroporater and 0.1 μm cuvettes. Colonies resulting from eachbacterial transformation were quantified, and plasmids from 20 coloniesfrom each muscle Hirt DNA sample were purified for analysis. It shouldbe noted that only circular forms carrying the Amp resistance gene andthe bacterial origin of replication from AV.GFP3ori are rescued bybacterial transformation (Duan et al., 1998). Control experimentsreconstituting 5×10¹⁰ viral DNA particles into uninfected muscleextracts prior to Hirt DNA preparation failed to give rise toreplication competent plasmids in the rescue assay (Duan et al., 1998).Additional controls in Duan et al. (1998) using AV.GFP3ori virus alsodemonstrated that linear double stranded and single stranded purifiedviral DNA genomes do not give rise to replication competent plasmidsfollowing transformation into E. coli.

Characterization of Encoded Genes in Rescued Circular Intermediates.

Several assays were used to characterize the extent of intermolecularrecombination between independent circular viral genomes by evaluatingthe number and type of encoded genes in rescued plasmids from Hirt DNAof muscles co-infected with AV.GFP3ori and AV.Alkphos. Initial analysisinvolved the bulk evaluation of 60 rescued plasmids (20 from each ofthree muscle samples for each time point) by dot blot hybridization ofmini-prep DNA with EGFP, Alkphos, and Amp ³²P-labeled DNA probes. Inthese studies, Amp hybridization served as a control to show that therewas a sufficient quantity of DNA for the analysis. The percentages ofAlkphos and/or GFP hybridizing plasmids were calculated by this methodfor each muscle sample. From this percentage, the total number ofplasmids hybridizing to each probe in the Hirt DNA sample was calculatedfrom the total CFU obtained in each transformation. In this analysis,each muscle sample was evaluated independently to determine the mean(+/−SEM) total Alkphos and/or GFP hybridizing plasmids. A secondevaluation involved the transfection of rescued plasmids into 293 cellsusing lipofectamine, followed by evaluation of GFP fluorescence andhistochemical staining for Alkphos. To confirm that GFP and Alkphosco-expressing plasmids were indeed clonal and that both genes wereencoded on the same plasmid, a selected group of five co-expressingplasmids were retransformed into E. coli and colonies were re-isolatedprior to repeating the transfection studies. In all cases, plasmidsco-expressing the two reporter genes remained clonal through thissubsequent re-isolation.

Structural Analysis of Concatamer rAAV Circular Intermediates.

To further characterize the nature of isolated circular intermediatesco-expressing both GFP and Alkphos transgenes, plasmid structure wasmapped by Southern blotting and restriction enzyme analysis. Thestructural of five co-expressing circular intermediate plasmids weredetermined by digestion with AhdI, HindIII, NotI, HindIII/NotI,ClaI/AseI, and/or SnaBI and Southern blotting was performed with³²P-labeled GFP, Alkphos, and ITR probes.

Results

Strategy for Characterizing Mechanisms of rAAV Circular IntermediateFormation.

Efficient circularization of rAAV genomes has been previouslydemonstrated to occur in muscle in a time dependent fashion (Example 2).Furthermore, the conversion of monomeric to multimeric circular rAAVintermediates occurred over time and was associated with long-termepisomal persistence of AAV genomes. High molecular weight AAV circulargenomes might form by either of the following two mechanisms, oneinvolving the replication of monomer structures and the other throughintermolecular recombination between independent monomers. A rescueassay was developed using two separate rAAV vectors, AV.GFP3ori andAV.Alkphos (FIG. 14A), which allowed for the identification ofindependent viral genomes through unique transgenes. In this assay,circular form genomes were rescued in bacteria by virtue of Amp/orisequences encoded in one of the two vectors (AV.GFP3ori). A method forcharacterizing the extent of intermolecular recombination betweenindependent circular rAAV genomes was shown in FIG. 14B.

Co-Expression of Independently Encoded rAAV Transgenes in MuscleMyofibers.

To confirm that myofibers can be co-infected at a high efficiency withthe two rAAV vectors, the tibialis anterior muscle of mice wasco-infected with 5×10⁹ DNA particles of both AV.GFP3ori and AV.Alkphos.At 14, 35, 80, and 120 days post-infection, muscles were harvested andanalyzed for transgene expression. Transgene expression from bothreporters was weak but clearly visible in 14 day muscle samples. By 80days post-infection, transgene expression was maximal and serialsections demonstrated expression of both Alkphos and GFP transgenes inoverlapping regions of the muscle (FIGS. 15A-C). At this time point,approximately 50% of the fibers in the tibialis muscle expressed bothtransgenes. To confirm that co-infection of myofibers occurred with thetwo independent vectors, co-localization studies were performed onmuscle sections by a serial staining procedure. These studies, depictedin FIG. 15D, demonstrate four classes of myofiber transgeneexpression: 1) GFP positive only, 2) Alkphos positive only, 3)GFP/Alkphos positive, and 4) no transgene expression. The largestfraction of myofibers expressed both GFP and Alkphos transgenes. Theseresults confirm that at the titers of virus used for infection,co-infection occurred in greater than 90% of transgene expressingmyofibers.

Rescue of Bi-Functional rAAV Circular Intermediates Increases Over Time.

To determine the extent of recombination between circular AAV genomes,circular form genomes were rescued as plasmids from low molecular weightHirt DNA of muscle tissue co-infected with AV.GFP3ori and AV.Alkphos.Following transformation of E. coli Sure cells with Hirt DNA purifiedfrom infected muscles, the total number of GFP and Alkphos hybridizingAmp resistant bacterial plasmids was quantitated for each time pointpost-infection (FIGS. 16A and B) (Duan et al., 1995), the abundance ofcircular AAV genomes rescued from AV.GFP3ori increased over time. Foreach muscle sample (three for each time point) twenty plasmid cloneswere evaluated for hybridization to GFP and Alkphos DNA probes and thetotal number of plasmids was back calculated from the total CFU for eachindividual muscle sample. FIG. 16B demonstrates the mean (+/−SEM, N=3)total plasmids that hybridized to GFP or GFP/Alkphos probes at each timepoint. At 14 days post-infection, GFP/Alkphos co-hybridizing plasmidswere never observed. In contrast, at time points after 35 days thepercentage of GFP/Alkphos co-hybridizing plasmids increased with timeand reached 33% by 120 days (FIG. 16C). Since bacterial plasmid rescuecan only occur through AV.GFP3ori genomes, this data suggests thatrecombination between independent Alkphos and GFP rAAV genomes takesplace over time. These results are consistent with studies describedhereinabove demonstrating a time dependent concatamerization of monomercircular rAAV genomes in muscle.

To evaluate the ability of circular intermediates to express encodedtransgenes, transient transfection studies were performed in 293 cellswith rescued circular intermediate plasmids (FIGS. 17A-C). Between85-90% of rescued plasmids hybridizing to GFP probes on slot blots alsoexpressed the GFP transgene in this transfection assay (FIG. 17D). Thepercentage of GFP expressing plasmids that also expressed Alkphos roseover time in concordance with the hybridization data (FIG. 17D).However, approximately 40-50% of plasmids which were hybridizationpositive for Alkphos did not express the Alkphos transgene. This mayrepresent recombinational deletion of the RSV promoter driving Alkphosexpression which occurred during concatamerization at sites near the 5′ITR. These results demonstrate that intermolecular recombination betweenAlkphos and GFP derived circular intermediates occurs as part of thetime dependent concatamerization process of rAAV in muscle. To confirmthat amplified plasmids stocks expressing both reporter genes wereactually clonal (i.e., one plasmid rather than two independent plasmidsresulting from contamination), a select number of bacterial clonesexpressing both transgenes were re-isolated and the transfection assayswere repeated. In all cases, plasmids expressing the two reporter genesremained clonal through two rounds of bacterial cloning. Hence, dualreporter expression was not due to contamination of independent GFP andAlkphos expressing plasmids.

Concatamerization of AAV Circular Intermediates Occurs Through UniformIntermolecular Recombination Between ITRs of Independent Viral Genomes.

To better understand the mechanisms of circular concatamer formation, adetailed structural analysis was performed of five bi-functionalcircular concatamers isolated from rAAV infected muscle samples. Aspreviously described for the AV.GFP3ori genome (Example 2), theconversion of monomeric circular AAV genomes to large multimericcircular concatamers with a predominant head-to-tail structure increasedwith time in muscle. To evaluate the structure of bi-functional circularconcatamers, restriction enzyme mapping and Southern blot analysis using³²P-labeled EGFP, Alkphos, and ITR probes was employed. Results fromfive analyzed plasmids demonstrated between 3-6 genomes within thesecircular concatamers. Two representative structures from 35 and 80 daytime points are shown in FIG. 18. Several interesting conclusions can bemade from this structural analysis. As described, head-to-tail orientedgenomes could be seen in all isolated concatamers. However, severalexamples of head-to-head and tail-to-tail genome combinations ofAV.Alkphos and AV.GFP3ori were also seen. Since head-to-head andtail-to-tail genome concatamers were never seen in muscles infected withAV.GFP3ori alone, there must be a selective disadvantage for bacterialreplication when ori sequences are in either of these conformations.However, since the AV.Alkphos genomes do not contain a bacterial originof replication, this orientation is permitted in chimeric concatamers.Second, noticeable deletions and/or loss of restriction sites close toITRs were noted (FIG. 17). It is not known whether deletions close tothe ITR are a common event in the concatamerization process, but if so,this could account for the fact that only 60% of GFP/Alkphos hybridizingcircular intermediates also expressed the Alkphos transgene.

Discussion

Concatamerization of rAAV genomes has long been recognized in integratedproviral genomes. Recently, the association of this concatamerizationprocess with the formation of high molecular circular genomes in musclehas suggested that this process may also be important in episomalpersistence. The findings described herein demonstrated rescue ofindependent viral genomes within the same circular concatamer,suggesting that this process of concatamerization occurs throughintermolecular recombination. Furthermore, at 14 days the predominantform of viral genome in muscle was circular monomers (Example 2), whichcorrelates with the results described above demonstrating only GFPexpression in rescued circular intermediates at this time point.Together with the fact that bi-functional rescued circular concatamersincrease with time, these results suggest that large concatamers form byrecombination of monomeric circular precursor genomes. Furthermore,since an alternative model of concatamerization by rolling circularreplication would be expected to yield only GFP expressing rescuedplasmids in this system, this mechanism does not appear responsible forconcatamerization.

Based on the structural analysis of these bi-functional circularintermediates, recombination between monomeric circular rAAV genomes islikely facilitated through ITR sequences. Directionality of thisrecombinational event does not appear to play a significant role, sincehead-to-tail, head-to-head, and tail-to-tail oriented intermolecularconcatamers were found. In addition, the extent to which recombinationwithin ITR repeat regions occurs in bacteria is presently unknown andmay account for the deletions and/or restriction site losses near ITRarrays. However, serial passaging of bi-functional circular AAV genomesin bacteria has suggested that the structure of these large concatamersis impressively stable in bacteria.

Intermolecular recombination of rAAV genomes to form single circularepisomes may be particularly useful for gene therapy. For example, largeregulatory elements and genes beyond the packaging capacity of rAAV maybecome linked after co-infecting tissue with two independent vectors(FIG. 19). This strategy could also involve splicing of transcripts fromvectors encoding two independent regions of a gene which are broughttogether to form an intact splicing unit by circular concatamerization.

For example, two independent vectors encoding two halves of the CFTRgene flanked by donor and acceptor splice site sequences are prepared.Expression of functional CFTR protein results after splicing of RNAtranscribed from a concatamerized genome comprising both halves of thegene in the sense orientation. One rAAV vector may comprise the first3.3 kb of the CFTR gene under the control of the RSV promoter and anin-frame splice donor site at the 3′ end of the CFTR cDNA. The secondrAAV vector encodes a splice acceptor intronic sequence, the 3′ 1.4 kbof the CFTR gene, and SV40 poly-adenylation sequences. To test forefficient splicing, a chimeric vector (pcDNA3.1CFTR-Donor/Acceptor) isintroduced to Xenopus oocytes by nuclear injection of the vector,followed by two electrode voltage (TEV) clamp recording functionalanalysis of CFTR (Jiang et al., 1998). mRNA transcripts are alsoanalyzed for correct splicing following transfection ofpcDNA3.1CFTR-Donor/Acceptor into MDCK cells. Polarized airway epithelialcells grown at the air-liquid interface are co-infected with the donorand acceptor CFTR AAV vectors CFTR gene expression in these cells isthen monitored by both immunofluorescent localization and functionalanalysis of short circuit currents (Smith et al., 1992; Smith et al.,1990). Hirt analyses of episomal AAV species are used to correlate theefficacy and persistence of CFTR gene expression with the formation ofAAV circular intermediates.

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All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain preferred embodiments thereof,and many details have been set forth for purposes of illustration, itwill be apparent to those skilled in the art that the invention issusceptible to additional embodiments and that certain of the detailsdescribed herein may be varied considerably without departing from thebasic principles of the invention.

1. An isolated and purified DNA molecule comprising at least one DNAsegment, a biologically active subunit or variant thereof, of a circularintermediate of adeno-associated virus, which DNA segment confersincreased episomal stability, persistence or abundance of the isolatedDNA molecule in a host cell.
 2. The DNA molecule of claim 1 in which theDNA segment comprises at least a portion of a 5′ inverted terminalrepeat of adeno-associated virus.
 3. The DNA molecule of claim 1 inwhich the DNA segment comprises at least a portion of a 3′-invertedterminal repeat of adeno-associated virus.
 4. The DNA molecule of claim1 which further comprises a marker or selectable gene.
 5. A plasmidcomprising the DNA molecule of claim
 1. 6. A gene transfer vector,comprising: a) at least one first DNA segment, a biologically activesubunit or variant thereof, of a circular intermediate ofadeno-associated virus, which DNA segment confers increased episomalstability or integration of the vector in a host cell; and b) a secondDNA segment comprising a gene.
 7. The vector of claim 6 in which thefirst DNA segment comprises at least about 550 bp of adeno-associatedvirus sequence.
 8. The vector of claim 6 in which the first DNA segmentcomprises at least about 400 bp of adeno-associated virus sequence.
 9. Atherapeutic gene transfer vector, comprising: a) at least one first DNAsegment, a biologically active subunit or variant thereof, of a circularintermediate of adeno-associated virus, which DNA segment confersincreased episomal stability or integration of the vector in a hostcell; and b) a second DNA segment comprising a gene encoding atherapeutically effective polypeptide.
 10. A method of delivering a geneto a cell comprising: contacting the cell with the vector of claim 6 or9.
 11. A composition for delivering a gene to a cell, comprising: thevector of claim 6 or 9 and a delivery vehicle.
 12. The composition ofclaim 11 wherein the delivery vehicle is a pharmaceutically acceptablecarrier.
 13. The composition of claim 11 wherein the delivery vehicle isa liposome.
 14. The DNA molecule of claim 1 which comprises concatamersof the circular intermediate.
 15. The DNA molecule of claim 1 in whichthe stability, persistence or abundance of the DNA in cells is enhancedby a DNA binding protein.
 16. The DNA molecule of claim 15 wherein theDNA binding protein is adenovirus E2a.
 17. A host cell comprising thevector of claim 6 or
 9. 18. A host cell comprising the DNA molecule ofclaim
 1. 19. An animal comprising the vector of claim 6 or
 9. 20. Theanimal of claim 19 which is not a human.
 21. A method of expressing agene product in the muscle tissue of an animal, which comprises:administering the vector of claim 6 or 9 to the muscle tissue of saidanimal in an amount effective to express the gene.
 22. The method ofclaim 21 wherein the vector is administered dissolved or suspended in aliquid pharmaceutically acceptable carrier.
 23. The method of claim 22wherein said liquid carrier comprises an aqueous solution.
 24. Themethod of claim 21 wherein said gene comprises a DNA segment encoding aprotein operably linked to a promoter operable in said muscle tissue.25. The method of claim 21 wherein said administering is byintramuscular injection.
 26. The method of claim 21 wherein saidadministering is by transdermal transport.
 27. The method of claim 21wherein said animal is a bird or mammal.
 28. The method of claim 1wherein said animal is a human.
 29. A method of expressing a gene in aeukaryotic cell, comprising: a) transfecting a eukaryotic host cellsusceptible to adenovirus infection with the vector of claim 6 or 9 anda recombinant adenovirus helper vector so as to form packaged viralparticles; and b) infecting a eukaryotic host cell with the viralparticles in an amount effective to detect expression of the gene.
 30. Acomposition comprising: a) a first adeno-associated virus vectorcomprising linked: i) a first DNA segment comprising a 5′-invertedterminal repeat of adeno-associated virus; ii) a second DNA segmentcomprising at least a portion of an open reading frame operably linkedto a promoter, wherein the DNA segment does not comprise the entire openreading frame; iii) a third DNA segment comprising a splice donor site;and iv) a fourth DNA segment comprising a 3′-inverted terminal repeat ofadeno-associated virus; and b) a second adeno-associated virus vectorcomprising linked: i) a first DNA segment comprising a 5′-invertedterminal repeat of adeno-associated virus; ii) a second DNA segmentcomprising a splice acceptor site; iii) a third DNA segment comprisingat least a portion of an open reading frame which together with the DNAsegment of (a)(ii) encodes a full-length polypeptide; and iv) a fourthDNA segment comprising a 3′-inverted terminal repeat of adeno-associatedvirus.
 31. The composition of claim 30 further comprising a deliveryvehicle.
 32. A method to transfer and express a polypeptide in a hostcell comprising contacting the host cell with the composition of claim30.
 33. A method to transfer and express a polypeptide in a host cellcomprising contacting the host cell with a first adeno-associated virusvector comprising linked: i) a first DNA segment comprising a5′-inverted terminal repeat of adeno-associated virus; ii) a second DNAsegment comprising at least a portion of an open reading frame operablylinked to a promoter, wherein the DNA segment does not comprise theentire open reading frame; iii) a third DNA segment comprising a splicedonor site; and iv) a fourth DNA segment comprising a 3′-invertedterminal repeat of adeno-associated virus.
 34. The method of claim 33wherein the host cell is further contacted with a secondadeno-associated virus vector comprising linked: i) a first DNA segmentcomprising a 5′-inverted terminal repeat of adeno-associated virus; ii)a second DNA segment comprising a splice acceptor site; iii) a third DNAsegment comprising at least a portion of an open reading frame whichtogether with the DNA segment of (a)(ii) encodes a full-lengthpolypeptide; and iv) a fourth DNA segment comprising a 3′-invertedterminal repeat of adeno-associated virus.
 35. The method of claim 32 or33 wherein the host cell is a lung epithelial cell, a muscle cell or aneuron.
 36. The method of claim 32 or 34 wherein the polypeptide is theCFTR polypeptide.
 37. A method of expressing a gene product in themuscle tissue of an animal, comprising contacting the muscle tissue withthe composition of claim 30 in an amount effective to express thepolypeptide.
 38. A method of expressing a gene product in the airwayepithelia of an animal, comprising contacting the airway epithelia withthe composition of claim 30 in an amount effective to express thepolypeptide.
 39. A method of expressing a gene product in the neurons ofan animal, comprising contacting the neurons with the composition ofclaim 30 in an amount effective to express the polypeptide.